Coinfection with multiple tick-borne pathogens

Alomst 1,000 species of ticks have been identified throughout the world. Many can transmit pathogens including protozoa, bacteria, and viruses. Infections with single tick-borne pathogens are considered common in people and domestic animals, while the prevalence of infections with multiple tick-borne pathogens is generally unknown. Both people and domestic animals are susceptible to coinfection with tick-borne pathogens, but, relative to single-pathogen infections, comparatively little is known about coinfections. In patients with clinical signs of one tick-borne disease, it is important to consider that they may be infected with multiple tick-borne pathogens. Coinfections may account for the diverse clinical signs some patients exhibit.1

Clinical signs previously attributable to single infections are now being reassessed in light of the awareness of coinfection. For example, epistaxis has not been observed in dogs with experimental Ehrlichia canis infection2-4 but has been reported in dogs seropositive for both E. canis and Bartonella vinsonii subsp berkhoffii.5 This suggests that dogs infected with E. canis and exhibiting epistaxis may be coinfected with Bartonella species.5 Response to treatment, and thus clinical outcome, may be influenced by the presence of additional tick-borne pathogens, so it behooves small-animal practitioners to consider the possibility of coinfections when they suspect or have confirmed that a patient is infected with a tick-borne pathogen.

IMPORTANT TICK VECTORS

In North America, six tick species are important vectors for the more common tick-borne diseases. They differ in their geographic distribution and the pathogens they can transmit (Table 1). Coinfections do not have to be acquired in a single geographic locale. Since some tick-borne infections can be chronic (e.g. ehrlichiosis), a patient's travel history is important in determining exposure to different tick species and tick-transmitted diseases.

Table 1. Tick Species That Are Important Disease Vectors and Their Associated Infective Organisms

MECHANISMS OF COINFECTION

Coinfection with tick-borne pathogens can occur through a number of mechanisms. A single tick species carrying multiple pathogens can transmit more than one organism to the same animal. Researchers in one study using polymerase chain reaction (PCR) testing to determine the prevalence of pathogens in Ixodes scapularis ticks in northern New Jersey documented that a single tick could carry Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia microti, and Bartonella species.6 In that study, 14% of individual ticks surveyed contained more than one pathogen. Other studies have reported lower rates of multiple-pathogen infection in individual ticks.7,8 From these studies, it appears that a small proportion of ticks could individually transmit multiple pathogens to susceptible hosts.

Ticks that are infected with one pathogen do not appear to have difficulty acquiring a second infection. Furthermore, these ticks can successfully transmit both pathogens into a susceptible host. In one study, about 60% of I. scapularis ticks dually infected with Borrelia burgdorferi and A. phagocytophilum successfully transferred both organisms to susceptible mice.9

In some situations, coinfection in the tick may increase the likelihood of transfer of a pathogen from a tick to a host. Ticks feeding on mice coinfected with B. burgdorferi and A. phagocytophilum contained significantly higher numbers of Borrelia spirochetes than did ticks feeding on mice solely infected with B. burgdorferi.10 Furthermore, 50% of ticks feeding on coinfected mice in this study contained evidence of A. phagocytophilum DNA, whereas A. phagocytophilum DNA was rarely detected in ticks feeding on mice infected solely with A. phagocytophilum.10 Although a relatively small percentage of ticks contain more than one pathogen, these ticks may be efficient at producing coinfections.

Coinfection can also be acquired from multiple tick species simultaneously feeding on a host and infecting it with multiple pathogens. This would be more likely in regions in which multiple tick species overlap geographically. Coinfection of animals can also result from the serial transmission of pathogens from multiple ticks at different times.

IMMUNOLOGIC ALTERATIONS AS RISK FACTORS FOR COINFECTION

A possible risk factor for coinfection is an alteration of the host immune responses caused by tick-borne pathogens. In one study, in vitro infection of a monocyte cell line with E. canis caused down-regulation of major histocompatibility complex class II receptors, suggesting presentation of ehrlichial or other antigens to CD4+ T cells; thus the immune responses dependent on such antigen presentation could be compromised.11 Bartonella vinsonii can induce immunosuppression by eliciting defects in monocyte phagocytosis, impairing antigen presentation within lymph nodes, and causing a decrease in circulating CD8+ T cells, which are important for cell-mediated immune responses.12 The effect of these immunologic changes on acquired immune responses could increase the risk of infection with other pathogens, including tick-borne agents, in a host that has compromised immune responses after infection with a single tick-borne pathogen.

Coinfection may have an even more profound effect on immune responses than do single infections. In mice, coinfection with Borreliaburgdorferi and A. phagocytophilum caused decreased activation of macrophages13 along with other alterations that could skew the immune response toward humoral immunity. These alterations would be of little benefit in eliminating an intracellular pathogen such as Ehrlichia species and could create a favorable environment for Ehrlichia species13 and other tick-borne agents to survive.

Whether immunity is altered in small animals infected with tick-borne pathogens is unclear. One study found that serum IgM, IgG, and IgA concentrations remained unchanged in young dogs after experimental infection with E. canis. In addition, the percentage of circulating CD4+ T cells was similar in infected and uninfected dogs, and functional defects of cell-mediated immunity were not observed.4 Factors that limit studies of the effects of infection with tick-borne pathogens on immune responses include the variability in vectors, differences in pathogenicity among strains, the effect of acute vs. chronic infection, and the wide variation in individual host immune responses. Not to be ignored is the fact that tick-host interaction may also alter the immune reaction. Tick salivary components or intestinal secretions introduced during feeding can shift the immunologic response to a humoral response in the surrounding skin and draining lymph nodes.14

PREVALENCE OF COINFECTION IN PEOPLE AND ANIMALS

Several reports document coinfection with tick-borne pathogens in people.15-19 The true prevalence of coinfection in people is unknown, but the medical literature suggests that coinfection is infrequent.15,18 A number of reports describe coinfections in veterinary patients,1,20-25 but there have been few epidemiologic studies on the prevalence of coinfections in dogs.

In one serologic survey of 277 dogs in Rhode Island, 21% were seropositive for two or more tick-transmitted pathogens, including Borrelia burgdorferi, Bartonella vinsonii, E. canis, and Rickettsia species.26 Coinfection with as many as six different tick-borne pathogens has been documented in dogs.1 In a study of dogs in Thailand, more than half of a population of dogs seroreactive to E. canis also had antibodies to Bartonella vinsonii subsp berkhoffii,20 leading another group of researchers5 to suggest that dogs exposed to E. canis in the United States and Thailand are also at risk of coinfection with B. vinsonii subsp berkhoffii. In another study, Bartonella species DNA was detected by PCR testing in seven of 12 dogs naturally infected with Ehrlichia species; five of 12 of these dogs were also seropositive for antibodies to B. vinsonii.21 Finally, about 26% of mice surveyed at United States military installations and training sites in Korea were found to be infected with multiple tick-borne pathogens.27

Collectively, the studies of coinfections in people, dogs, and other species emphasize the fact that evidence of naturally occurring coinfections in animals can be found when efforts are made to look for them.

WHEN TO SUSPECT COINFECTION

Susceptibility to coinfection may be affected by factors such as geography, travel, husbandry, host immune status, and the use of preventive drugs and prophylactic therapy. Knowing the common tick species and pathogens within a practice's geographic area, as well as a patient's travel history, is paramount if coinfection is suspected. Suspicion for coinfection should be high if a patient has had frequent exposure to vectors or reservoir hosts or if there is a high prevalence of disease in a practice area.

In one report, factors associated with a high rate of coinfection with Ehrlichia species, Anaplasma species, Babesiacanis, Bartonella vinsonii, and Rickettsia rickettsii in a Walker hound kennel included cohabitation; frequent exposure to ticks, mice, and rodents in the vicinity of the kennel; and frequent travel to wooded areas.1

Coinfection should also be suspected if a patient is more clinically ill than would be expected based on infection with a single pathogen or if a patient has an atypical clinical presentation. Histopathologic examination of joints from mice experimentally coinfected with Borrelia burgdorferi and A. phagocytophilum showed more severe arthritis at two weeks after infection than did mice infected with B. burgdorferi alone, while mice solely infected with A. phagocytophilum did not develop arthritis.10 In people, worse clinicopathologic changes may result from concurrent borreliosis and babesiosis18,19 or concurrent borreliosis and A. phagocytophilum infection.10,18 Although there is not yet direct evidence that more severe clinical disease occurs in coinfected companion animals, these studies in other species may serve as a valuable precedent when evaluating veterinary patients.

Yet another situation in which to suspect coinfection is if a patient has either a delayed response or no response to appropriate therapy directed at a single pathogen. For example, in a patient with E. canis infection, if platelet counts do not increase within seven days of therapy, another mechanism for thrombocytopenia should be suspected, such as immune-mediated destruction or coinfection with Babesia or Bartonella species.28 In one report, a dog coinfected with Bartonella vinsonii subsp berkhoffii and Babesia canis had persistent thrombocytopenia until both infections had been treated.23

Another laboratory indicator supporting coinfection may be a positive antinuclear antibody (ANA) test result. One study demonstrated that dogs that were seroreactive to multiple tick-borne pathogens were more likely to have positive ANA results than were dogs from the same geographic region that were seroreactive to only one antigen.25

DIAGNOSING A COINFECTION

A diagnosis of infection by a single tick-borne pathogen is typically made by using a combination of physical examination findings, clinicopathologic abnormalities, and results from pathogen-specific tests such as direct microscopic visualization, serologic testing, immunoblotting, immunodetection of organisms in blood or infected tissue, microbial culture, or PCR testing.29 The same strategies apply to diagnosing coinfections.

Hematologic abnormalities

One of the most common hematologic abnormalities associated with tick-transmitted diseases in dogs is thrombocytopenia.28 In fact, because of the high incidence of thrombocytopenia in dogs infected with E. canis, platelet counts have been suggested as a screening test for this disease in endemic regions. One study demonstrated that only one of 71 nonthrombocytopenic dogs has positive results for E. canis,30 suggesting that a platelet count as a screening test has a high positive predictive value but low negative predictive value. However, because other tick-transmitted diseases were not excluded, the contribution of coinfection to the thrombocytopenia observed cannot be assessed.

Cytology

Cytologic examination of blood smears and joint fluid is a simple diagnostic tool that is generally underused and may help raise the suspicion for a coinfection. Cytologic examination has moderate sensitivity for diagnosing acute, but not chronic, E. canis infection. A study in dogs comparing the sensitivity of buffy coat, peripheral blood, lymph node, and bone marrow evaluation in acute E. canis infection determined that the combination of a buffy coat smear and lymph node evaluation had a sensitivity of 74%.31 In contrast, only about 10% of chronically infected dogs had cytologic evidence in bone marrow.32

In a study of experimental Hepatozoon canis infection, four of five puppies inoculated by ingestion of Rhipicephalus sanguineus had gametocytes evident on a blood smear, and the fifth dog had gametocytes in aspirates of the spleen and bone marrow.33 Dogs naturally and experimentally infected with A. phagocytophilum have exhibited inclusion bodies within neutrophils.34,35 Babesia species parasitemia is often low in peripheral blood smears; however, blood collected from peripheral capillary beds of the ears or nail beds or smears made from cells near the buffy coat often yield higher numbers of organisms.36 In addition, cells examined in the periphery of the smear contain more intracellular organisms.36 Observing inclusions within different cell types should alert practitioners to the existence of concurrent infections.

The obvious disadvantages of a cytologic examination are that it is labor-intensive and time-consuming and it requires thorough examination of many fields and the use of appropriate stains (Giemsa). Nonetheless, it is accessible to most clinicians. With the increasing use of in-house automated hematology analyzers in many small-animal practices, blood smear examination is regrettably too frequently omitted in the hematologic assessment of ill patients. Cell tropism is summarized in Table 2.

Table 2. Intracellular Inclusions

Serologic testing

Serologic assays, predominantly immunofluorescent antibody (IFA) tests, have been the mainstay of diagnosis for most tick-borne infections. However, serologic testing has limited ability to identify acute infection or to differentiate infection from prior exposure or vaccination (e.g. Lyme disease). A high degree of serologic cross-reactivity exists among species within the same genogroup; thus, for example, seroreactivity to E. canis may in fact represent exposure to Ehrlichia chaffeensis or Ehrlichia ewingii. Serologic cross-reactivity occurs among other pathogens.29 Patterns of cross-reactivity are summarized in Table 3.

Table 3. IFA Cross-reactivity

Further complicating serologic testing is the fact that chronic, but not acute, infection with E. canis causes cross-reactivity with A. phagocytophilum. In one study, six of six dogs experimentally infected with E. canis became seropositive for A. phagocytophilum within 150 days after infection with E. canis.37 Infection with A. phagocytophilum can also cause cross-reactivity with E. canis.21 It has been proposed that the appearance of A. phagocytophilum genogroup antigens in dogs infected with E. canis may indicate persistent E. canis organisms, and, in treated cases, their appearance may be a serologic marker of treatment failure.37

The development of enzyme-linked immunosorbent assays (ELISAs) for identification of ehrlichiosis and borreliosis has allowed for quicker in-house testing. Comparing a diagnosis of ehrlichiosis based on positive results of the ELISA-based Snap 3Dx Test (IDEXX Laboratories) with IFA showed an overall agreement of 91% (61/67) in experimentally and naturally infected animals, with all disagreement occurring in samples found to have IFA titers of 1:320 or less.38 Sensitivity and specificity of the Snap 3Dx Test were 0.71 and 1.00, respectively.38 In a later study comparing the Snap 3Dx Test with a commercial IFA, similar results were found. Stored serum samples known to be nonreactive, low-reactive (titer 80 to 160), medium-reactive (titer 320 to 2,560), and highly reactive (titer 5,120 to > 20,480) by IFA were consistent with ELISA results in the nonreactive, medium-reactive, and highly reactive groups.39 Discordance was only identified in the low-reactive group, indicating ELISA sensitivity is poor for this group; however, the authors postulated that poor specificity of the IFA could result in this discordance as well.39 Unfortunately, in each study, naturally infected cases were not evaluated with PCR testing to exclude coinfection or assess cross-reactivity. Because ELISAs give clinicians no information about titers, the tests have somewhat limited use in assessing therapy for which decreases in serum titers would be considered a positive response to treatment. Although the IFA is subject to cross-reactivity, because of better sensitivity as compared with ELISA, it remains the test of choice for diagnosing ehrlichiosis.

The Snap 3Dx Test detection of borreliosis was highly sensitive and specific.40 In addition, the ELISA was able to distinguish vaccinated from unvaccinated individuals.40 Disadvantages of this test relate to the high prevalence of exposure to Borrelia burgdorferi in the population in addition to limitations that parallel those described above for detecting ehrlichiosis. Despite these disadvantages, the Snap 3Dx Test is a viable option in place of IFA for diagnosing borreliosis when there is an appropriate history and physical and laboratory abnormalities.

PCR testing

PCR testing is useful for detecting and differentiating among tick-borne diseases. When serologic testing is inconclusive, PCR testing may help confirm a diagnosis. However, PCR testing generally should be an adjunct and not the sole means of detecting infection. PCR results do not always correlate well with serology, and, in most instances, infection is documented with serology and not PCR testing.

In a study evaluating serologic (IFA) and PCR evidence of coinfection in dogs from Thailand, only 10 of 36 dogs seroreactive to E. canis had positive PCR test results.20 The authors of that study speculated that a negative PCR test result in a seropositive dog could be expected after immunologic or therapeutic cure or with low circulating numbers of organisms. In a similar study, 13 of 19 dogs at one institution and five of 19 dogs at another institution that were seroreactive by IFA had positive PCR test results.32

An advantage that PCR testing has over other modalities is the ability to identify the species causing infection, which is particularly helpful for pathogens for which there is extensive serologic cross-reactivity. Positive PCR results also add support to a diagnosis of an active infection by organisms such as Babesia or Borrelia species, to which dogs may be exposed and, thus, seropositive for but for which seroreactivity does not always equate to active infection. Detection of DNA for a specific tick-borne agent in a patient seropositive for the same organism supports a diagnosis of active infection when there are other supportive findings (e.g. from history, physical examination, other diagnostic tests). Another advantage of PCR testing is that it can be used to look for evidence of infection by one or more of a group of related organisms (e.g. Ehrlichia species) by using primers specific for the genus of interest. A PCR test result positive for Ehrlichia species could then suggest the need for additional PCR tests to identify the species present.

Limitations of PCR testing include limited availability, expense, inconsistent quality control, and lack of standardization among laboratories. Diagnostic PCR assays are improving with the implementation of real-time and nested PCR strategies and, as a result, are becoming more sensitive and specific.41,42

POTENTIAL INFLUENCE OF COINFECTION ON TREATMENT AND CLINICAL OUTCOME

Whenever possible, the goal when evaluating an ill patient is to make a diagnosis so that you can implement specific therapy. For patients with tick-transmitted diseases, failure to consider coinfection can lead to inappropriate and ineffective therapy, possibly prolonging the course of clinical illness. In the case cited earlier of the dog with bartonellosis and babesiosis,23 the dog's illness extended over five months until babesiosis was identified and treated.

Although many tick-borne infections are successfully treated with tetracycline and related antibiotics, this antimicrobial choice is not appropriate for all tick-transmitted diseases. While a clearly superior treatment protocol has not been established for Babesia canis infection, tetracyclines and imidocarb dipropionate are both effective.43 An optimal protocol for treating infection with Bartonella species has not been determined either but may include, in addition to tetracyclines, empirical treatment with antimicrobials such as enrofloxacin, azithromycin, and trimethroprim-sulfamethoxazole.5,44-49 Thus, patients with coinfections could require therapy with several different antimicrobials to effectively treat all pathogens.

Treatment and subsequent clinical recovery does not guarantee cure or immunity. It is important to note that dogs can become persistently infected or repeatedly reinfected with some of the tick-borne pathogens in endemic areas. Some dogs may have persistence of antibody titers after treatment, but the persistence of antibody reactivity after anti-rickettsial drug therapy does not necessarily equate with treatment failure. Dogs can have persistently elevated antibody titers for up to 36 months after treatment despite resolution of clinical signs and laboratory abnormalities.50 Hence antibody detection can be misleading in assessing treatment efficacy. PCR testing may help in assessing treatment efficacy because it may be able to differentiate between animals persistently infected and those that have persistent antibody titers despite successful treatment.1,51

CONCLUSION

Coinfection may account for some of the variations seen in clinical presentation, pathogenicity, and therapeutic response in patients with tick-borne infections. Evidence indicates that coinfections are found in some patients if an effort is made to identify them. Clinicians must use a combination of geography, clinical findings, and laboratory findings to help determine whether coinfection is present and decide which specific pathogens to test for. Knowing the advantages and limitations of each test can help clinicians efficiently use the arsenal of diagnostics available today. The use of molecular diagnostic assays such as PCR testing will help in documenting coinfections and contribute to a better understanding of the incidence of coinfections as well as our understanding of the clinical syndromes caused by coinfections.

Adam Mordecai, DVM

Erick Spencer, DVM

Rance K. Sellon, DVM, PhD, DACVIM

Department of Veterinary Clinical Sciences

College of Veterinary Medicine

Washington State University

Pullman, WA 99164-7060

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