Clinicians, Causation and Infectious Disease Edward B. Breitschwerdt, DVM Professor of Medicine and Infectious Diseases College of Veterinary Medicine North Carolina State University Raleigh, NC

It is increasingly obvious that microorganisms, including Anaplasma,Babesia,Bartonella, Borrelia, Chlamydia, Ehrlichia, Leishmania, Mycoplasma species and retroviruses can persist in the blood or other tissues of animals for protracted periods of time. The same evolutionary adaptations that facilitate organism persistence complicate the clinical confirmation of causation. This is particularly true when assessing diagnostic test results for an individual patient residing in a highly endemic area for vector borne infections. Therefore, clinicians need to recognize that the mammalian body functions as a component of numerous complex ecosystems, which facilitates the evolutionary perpetuation of bacteria, protozoa and viruses. In practical terms, this means that the detection of antibodies, antigens, DNA, RNA or the culture of microorganisms does not directly support a cause and effect relationship between detection of the microorganism and disease manifestations found in an individual patient.

It is also increasingly obvious that disease expression is frequently multi-factorial. When multiple factors simultaneously induce chronic changes in structural, biochemical and physiological processes over a protracted period of time, disease expression can be highly variable, even among a specified population. Although epidemiological studies attempt to control for variation in study populations, frequently, only a limited number of factors can be simultaneously assessed or controlled for in the context of the study design. The influence of unknown or undetected infectious agents can not be controlled for in the context of study design. An example was the discovery of several Helicobacter species that infect rodents used to experimentally determine the oncogenic potential of various chemicals evaluated by the National Institute for Environmental Health Sciences and other testing agencies. Retrospective analysis found that the presence or absence of Helicobacter species, such as H. hepaticus, had unknowingly influenced the experimental results. In certain instances Helicobacter-infected rodents were more likely to develop cancer.

As the factors that influence disease expression can differ significantly among individuals within a given population, the clinician should anticipate a broad spectrum of clinical, hematological or biochemical abnormalities when focusing diagnostic emphasis on a single factor or test result. As an example, diverse disease manifestations including epistaxis, immune-mediated hemolytic anemia, immune mediated thrombocytopenia, glomerulonephritis, polyarthritis and myelofibrosis, have been historically attributed to Ehrlichia canis infection in dogs. For a variety of reasons, it is increasingly clear that canine ehrlichiosis is a disease complex that is influenced by the infecting Ehrlichia species, the host immune response, co- infection with other vector borne or non-vector borne pathogens and perhaps numerous other factors, yet to be clarified. In addition to E. canis, other species (E. chaffeensis E. ruminatum and E. ewingii) can cause ehrlichiosis in dogs and in people. In conjunction with numerous highly variable host factors, the infecting Ehrlichia species can influence disease expression. For example, polyarthritis is most frequently associated with E. ewingii infection in dogs, than with E. canis or E. chaffensis infection. It is also possible that genetic variation in organism virulence can influence disease severity. For example, although other unknown factors may be involved in disease expression, dogs infected with E. canis in Greece are more likely to develop severe bone marrow suppression that is frequently not therapeutically responsive to doxycycline. Both of these observations are in direct contrast with the overall experience with E. canis infections in the United States (i.e. pancytopenia is rare and most dogs, despite chronic illness, respond hematologically following administration of doxycycline). In addition, recent data indicates that the common evolutionary history of Anaplasma, Bartonella and Ehrlichia species has resulted in complex polymicrobial, interactions that influence the pathophysiology of disease expression in animal and human patients. For the clinician, confirming active infection caused by a single tick borne pathogen can be challenging, particularly when evaluating chronic as compared to acute illness. Seroconversion, culture and PCR can all be used to support causation in an acutely infected indivdual. Using these same diagnostic modalities, the microbiological confirmation of polymicrobial tick borne infections in a chronically ill patient can be extremely difficult or in certain instances impossible.

It has been recognized for some time that A. phagocytophilum can induce disease in cats, dogs, horses and human beings and can infect numerous other wild animal species that serve as reservoir hosts. Similarly, E. canis, E. chaffeensis, E. ewingii and seemingly E. ruminatum organisms, which are transmitted by a wide spectrum of tick species, can infect both dogs and people. Bartonella vinsonii (berkhoffii), initially isolated from a dog with endocarditis in our laboratory, was subsequently isolated from a human with endocarditis in Europe. Infection with Bartonella henselae, identified in the early 1990’s as the predominant cause of cat scratch disease (CSD) and bacillary angiomatosis and peliosis hepatis in immunocompromised individuals, is now known to be a much more prevalent infection in dogs than was previously recognized. Recently, using a novel culture approach, we have isolated B. henselae from dogs that lacked detectable Bartonella antibodies by indirect immunofluorescent antibody (IFA) testing and were PCR negative using a highly sensitive real time PCR assay. This illustrates the limitations of both serology and RT-PCR for the confirmation of B. henselae bacteremia in dogs and potentially other animals.

In the dog, the medical implications of polymicrobial infection with two or more tick-transmitted evolutionary partners are essentially unknown. Because Anaplasma, Babesia Ehrlichia, Leishmania and Mycoplasma spp could be visualized during microscopic examination of blood smears or tissue aspirates, the discovery of these organisms predates the discovery of pathogenic Bartonella spp., which are not visualized on stained blood smears. Based upon serology, there is an association between exposure to E. canis and B. vinsonii berkhoffii and a similar association between A. phagocytophilum and B. henselae exposure. Presumably, these associations are related to co-transmission by Rhipicephalus sanguineus or Ixodes scapularis, pacificus and ricinus, respectively. Recently, using PCR amplification of organism specific DNA, researchers found a higher prevalence of Bartonella spp. DNA than B. burgdorferi DNA in I. scapularis ticks collected in New Jersey. Collectively, evolving evidence suggests that clinicians should pursue a diagnosis of concurrent bartonellosis in dogs with anaplasmosis, babesiosis or ehrlichiosis. It also suggests that the world literature related to canine ehrlichiosis should be reassessed, based upon the high frequency of Bartonella spp. co-infection in E. canis infected dogs. These evolutionary relationships are also supported by molecular evidence of polymicrobial infection in dogs and other animals, including man. After isolating E. chaffeensis from a captive lemur, we determined that the culture was concurrently infected with B. henselae, despite our inability to visualize this organism with stains used to visualize E chaffeensis. This isolation result suggests that during experimental infection studies using blood transfusion from an E. canis infected dog or tissue culture grown organisms as inoculums, investigators may have unknowingly transmitted Anaplasma, Bartonella and Ehrlichia spp, which would have obvious implications for disease expression and the interpretation of the experimental results. This may in part explain the substantial variation in the severity of clinical and hematological abnormalities reported following experimental infection of dogs with E. canis.

Geographic variation in the prevalence of tick-transmitted pathogens presents an important challenge for veterinary clinicians. For example, in the northeastern United States, I. scapularis can transmit A. phagocytophilum B. burgdorferi, Babesia microti, and Bartonella vinsonii (arupensis). In the southeastern United States, dogs are more frequently exposed to Dermacentor variabilis, Amblyomma americanum and R. sanguineus, which could result in infection with Anaplasma platys, B. canis, B. vinsonii (berkhoffii), E. canis, E. ewingii, E. chaffeensis, R. rickettsii, and other less pathogenic spotted fever group rickettsiae, such as R. montana and R. rhipicephali. Therefore, it has become increasingly obvious that ticks in different geographic regions or localities can transmit different pathogens. As many tick-transmitted infections result in a prolonged sub clinical course, a dog or cat might be infected in an endemic area, where veterinarians are very familiar with the disease manifestations. Unfortunately, the dog may become ill months to years later, after moving to an area in which the disease is not endemic and where veterinarians are far less familiar with the disease manifestations. This emphasizes the importance of obtaining a travel history for all sick animals, when a vector transmitted infectious disease is a diagnostic consideration.

Determining the relative role of each factor that contributes to disease causation is currently not possible because diagnostic screening tests that can simultaneously assess the genetic, immunological, nutritional and toxicological status of a patient are highly insensitive or generally unavailable. In addition, assessing the infection status of a patient is also challenging, due to poor test sensitivity or specificity. Although clinicians tend to accept the reliability of sensitivity and specificity data, as reported by diagnostic laboratories or independent researchers, “beauty”, or in this instance diagnostic accuracy, is frequently in the eye of he beholder. An excellent contemporary example relates to the putative role of Chlamydia pneumoniae in the development of human atherosclerosis. Chlamydia pneumoniae was first described as a human pulmonary pathogen in 1987. During the past 15 years, numerous studies have attempted to determine if there is a causative relationship between infection with C. pneumoniae and atherosclerosis. Although C. pneumoniae has been detected in atherosclerotic lesions by electron microscopy, immunohistochemistry, in vitro cultivation, PCR or in situ hybirdization, the controversy regarding causation continues unabated. Similar controversy exists in veterinary medicine regarding the role of B. burgdorferi as a cause of acute glomerular injury or chronic arthritis in dogs and the role of B. henselae as a cause of gingivitis and stomatitis in cats. A problem that is common among studies that address causation in regard to these three infectious agents relates to the hypothesis that a single, highly adapted organism can independently induce complex disease expression. Contemporary researchers are questioning the appropriateness of Koch’s postulates for determining the relative contribution of one or more infectious agents to the development of chronic disease. To effectively define the pathophysiological complexities of chronic disease expression, infectious disease researchers will need to design studies in conjunction with epidemiologists, immunologists, microbiologists, nutritionists and toxicologists. In the interim, clinicians will continue to make important medical decisions for their patients, with less than adequate data.

Selected References: Hailey JR, Haseman JK, Bucher JR, Radovsky AE, Malarkey DE, Miller RT, Nyska A, Maronpot RR: Impact of Helicobacter hepaticus infection in B6C3F1 mice from twelve National Toxicology Program two-year carcinogenesis studies. Toxicol Pathol. 26:602-11, 1998. Mylonakis ME, AF Koutinas, EB Breitschwerdt, BC Hegarty, CD Billinis, LS Leontides, VS Kontos: Chronic canine Ehrlichiosis (Ehrlichia canis): A retrospective study of 19 natural cases. J Am Anim Hosp Assoc 40:174-184; 2004. Kordick SK, EB Breitschwerdt, BC Hegarty, KL Southwick, CM Colitz, SI Hancock, JM Bradley, R Rumbough, JT McPherson, JN MacCormack: Coinfection with multiple tick-borne pathogens in a Walker Hound Kennel in North Carolina. J Clin Microbiol 37: 2631–2638, 1999. Breitschwerdt EB, CE Atkins, TT Brown, DL Kordick, PS Snyder: Bartonella vinsonii supsp. Berkhoffi and related members of the alpha subdivision of the Proteobacteria in dogs with cardiac arrhythmias, endocarditis, or myocarditis. J Clin Microbiol 37: 3618–3626, 1999. Lappin MR, EB Breitschwerdt, WA Jensen, B Dunnigan, J-Y Rha, CR Williams, M Brewer, M Fall: Molecular and serologic evidence of Anaplasma phagocytophilum infection in cats in North America. J Am Vet Med Assoc 225(6):893-896; 2004. Maggi RG, Duncan A, Breitschwerdt EB: A novel chemically modified liquid medium that will support the growth of seven Bartonella species. J. Clin Microbiol In press. Adelson ME, Rao R-VS, RC. Tilton RC, Kimberly Cabets K, Eskow E, Fein L, Occi JL, Mordechai E: Prevalence of Borrelia burgdorferi, Bartonella spp., Babesia microti, and Anaplasma phagocytophila in Ixodes scapularis Ticks Collected in Northern New Jersey J. Clin Microbiol 42: 2799-2801, 2004. Williams CV, JL Van Steenhouse, JM Bradley, SI Hancock, BC Hegarty, EB Breitschwerdt: Naturally occurruing Ehrlichia chafeenis infection in two prosimian primate species: ring-tailed lemures (Lemur catta) and ruffed lemurs (Varecia variegata). Emerg Infect Dis 8:1497-1500; 2002. Ieven MM, VY Hoymans. Involvement of Chlamydia pneumoniae in atherosclerosis: More evidence for lack of evidence. J Clin Microbiol 43: 19-24, 2005.