2018

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Edward B. Breitschwerdt, BS, DVM, DACVIM North Carolina State University, College of Veterinary Medicine

ebbreits@ncsu.edu

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SEROLOGICAL AND MOLECULAR DIAGNOSIS OF INFECTIOUS DISEASES: Something old and something new. Part I

Edward B. Breitschwerdt DVM Diplomate ACVIM

Center for Comparative Medicine and Translational Research Department of Clinical Sciences, College of Veterinary Medicine

North Carolina State University, Raleigh, NC 27606

INFECTIOUS DISEASES DIAGNOSIS: A CLINICAL EXAMPLE: I will use as an example, a consultation from a practice owner in Iowa (one of

yesterday’s consults) to illustrate the complexities of infectious diseases diagnosis in the practice environment. The veterinarian contacted me by e-mail because one of his “ younger associates” had evaluated a dog that initially presented for acute onset of weakness and mild anemia (PCV 27%). Following “symptomatic therapy” the hematocrit normalized for several weeks (rechecked at least 4 times), however, on a subsequent recheck, the hematocrit was 12%, immune-mediated hemolytic anemia was diagnosed, and the dog was immediately referred to Iowa State University, College of Veterinary Medicine. A tick borne pathogen panel was sent to the NCSU-VBDDL (Vector Borne Diseases Diagnostic Laboratory), but the dog died before test results were available. Babesia canis antibodies (titer 1: 64) were detected by IFA testing in our laboratory and additional review of the dog’s history determined that the dog had traveled to Arkansas. The client was very unhappy and the veterinarian was seeking information related to canine babesiosis in dogs in Iowa. IMPLICATIONS FOR VETERINARY MEDICINE: 1. A diagnosis was made (perhaps), but too late to benefit the patient. 2. Serological evidence of exposure does not confirm active infection. 3. Because of Adam Birkenheuer (and others) babesiosis is an emerging canine

infectious disease in the United States. 4. Babesiosis is a definitive cause of immune-mediated hemolytic anemia in dogs. 5. Based upon more recent obvservations, both visualization of Babesia organisms on

blood smears and serological testing are insensitive diagnostic tests, as compared to PCR.

6. Arkansas is highly endemic for Babesia canis. Iowa is not endemic for B. canis. 7. Dogs, owners and other animals travel farther and more frequently than at any other

time in history (i.e. SARS, Monkey Pox, West Nile Virus to name a few). 8. It takes only one brown dog tick to transmit this organism. This tick is usually found

in kennels (boarding), homes (visiting friends) or veterinary hospitals (heaven forbid). 9. Even the best parasitology and medicine courses may not have prepared this young

graduate to deal with this patient presentation. (To be sure!) 10. A complete medical history will always serve as the foundation of optimal medical

care. 11. There is not a Center for Disease Control (CDC) or Infectious Diseases Society of

America that tracks companion animal infectious diseases, therefore there is no data base to address regional or national concerns.

SO WHAT ARE THE SOLUTIONS?

Clearly, in the context of infectious diseases, clinicians need diagnostic tests that allow for the simultaneous detection of common, uncommon and unknown infectious agents. This seemingly impossible option is becoming much more probable with the advent of molecular biology and highly sensitive and specific DNA or RNA detection

systems. Although molecular diagnostics have and will continue to have limitations, their incorporation into the practice of veterinary medicine will occur at an increasingly accelerated rate. Efforts to develop highly sensitive pathogen detection methodologies for bioterrorism, will in many instances have direct applicability to clinical medicine. Bioterrorism and substantial federal funding are responsible in part for the accelerated development of technology that veterinarians will use in the future. IMMUNOLOGICAL IMBALANCE AND INFECTION

Infection is defined as the invasion and multiplication of microorganisms in body tissues. Invasion infers that infection alters the immunological balance within an individual (i.e. invasion causes an immunological battle that can range in severity from a skirmish to all out war). It is increasingly obvious that this definition lacks clinical utility in regard to organisms that can achieve long term intracellular or extracellular persistence within the host. In these instances, it appears that both the host and the organism work in concert to maintain a state of immunological balance. For example, Helicobacter pylori can be found within the gastric lumen of nearly 100% of people in developing nations and 30-50% of people in developed nations. Most individuals are infected during childhood and remain infected for the remainder of their life. Despite the large number of infected individuals and the presence of chronic superficial gastritis in all H pylori infected individuals, only a small percentage of the population develops clinically apparent disease manifestations, which can include gastric ulcers, gastric MALT (mucosa associated lymphoid tissue) lymphoma or gastric adenocarcinoma. In this example, as well as others, disease expression is most likely multifactorial. It is very obvious that host genetics, microbial genetics, nutrition and exposure to toxic chemicals have a major influence on disease expression. CONCURRENT INFECTIONS AND DISEASE EXPRESSION

If one were to assume that viruses, bacteria and protozoa had to communicate with each other in order to sustain life within a complex ecosystem, i.e. the mammalian body, then it would follow that these organisms should manipulate the highly developed and complex host immune responses in a coordinated and synergistic manner. For example, while attempting to clarify an increase in unexplained deaths in a Walker hound kennel, we were able to amplify DNA of up to six different organisms (i.e. 6 different species from 4 different genera) from an EDTA-anti-coagulated blood sample, obtained at a single point in time. What are the potential clinical implications of this observation? First, dogs with extensive tick, flea and louse exposure can be simultaneously infected (based upon detection of DNA) with multiple organisms. Secondly, these dogs were working dogs that were “genetically” capable of compensating to a substantial degree for simultaneous infection with bacteria, protozoa and rickettsiae. However, over half of the dogs in the kennel had ocular abnormalities, including anterior uveitis, ocular hemorrhage or retinitis, generally accompanied by thrombocytopenia, mild anemia and hyperglobulinemia. Although considered by the owner’s to be functional working dogs for deer hunting, it is likely that these dogs were no longer in a state of immunological balance. It is also likely that the imbalance was multi-factorial and that other factors such as nutrition and toxicity contributed to the development of ocular, hematological, cardiovascular and renal pathology. Finally, if the limited spectrum of PCR testing utilized in our laboratory was able to detect six different species in a single dog, it is highly likely that other organisms were present within this ecosystem (the blood of kennel dogs) for which no testing was performed. One possible conclusion, derived from recent research, is that the mammalian body is an ecosystem that encompasses a highly developed immune system that interacts constantly with

microorganisms on the skin, mucosal surfaces and within blood and other tissues. Imbalance in the system is rarely due to a singular factor or event and rarely precipitated by a single microorganism. SEROLOGY AND INFECTIOUS DISEASES DIAGNOSIS Historically, serology has been the mainstay of diagnosis for difficult to isolate bacteria, protozoa and viruses. Serology still has an important role in infectious diseases diagnosis and ideally should be used in conjunction with isolation or molecular diagnostic approaches. Unfortunately, the isolation of many bacteria, protozoa or viruses is technically challenging, limited to research settings, and requires prolong incubations periods (weeks to months) for successful isolation. For an acute disease process, such as Leptospirosis, Rocky Mountain spotted fever or anaplasmosis, documentation of seroconversion can be used to confirm a diagnosis. Seroconversion is defined in most instances as an at least a 4-fold increase in antibody titer between acute and convalescent test samples. Documentation of seroconversion requires a testing modality, such as indirect fluorescent antibody or kinetic ELISA. that determines the quantity of antibodies in the respective patient samples. When a clinician plans to document seroconversion, an initial patient sample should be obtained as early in the course of illness as possible. As the antibody titer in the initial sample may be low (below the laboratory diagnostic cut off) or not detectable, this sample can be stored for 2-3 weeks in a refrigerator or freezer until the convalescent sample is obtained. Although seroconversion can confirm a specific diagnosis, which may be very important if the infectious agent is zoonotic, documenting seroconversion does not help the clinician in selecting a treatment approach for the patient. For acute infections, treatment, such as doxcycline for acute anaplasmosis, ehrlichiosis or Rocky Mountain spotted fever, must be initiated prior to diagnostic confirmation of the disease process. When antibodies are detecting in a healthy animal or in association with a chronic infection, the presence of antibodies supports prior exposure and immunological recognition of the infecting organism, but does not confirm that the animal (healthy or sick) is actively infected with the organism for which antibodies were detected. Although treatment decisions can and frequently are based upon antibody detection, this approach should be used with caution, particularly when antibodies are detected using screening tests in healthy pets. In the context of vector-borne infectious diseases, such as anaplasmosis, babesiosis, borreliosis (Lyme Disease), ehrlichiosis, leishmaniasis and rickettsioses, exposure can be extensive in various animal populations. In some instances, the organism is eliminated following infection by the host immune response, which means that active infection no longer exists and treatment directed at therapeutic elimination of the organism is not warranted. In other instances, a state of immunological balance is established between the infecting organism and the host immune response (the state of premunition). Although persistently infected these animals can be outwardly and clinically healthy and may or may not have hematological or biochemical evidence of disease. In many instances, data is not available to define the long term consequences of these occult infections and the number of animals that will progress from a clinically healthy state to active disease. I have become somewhat fond of saying to our internal medicine residents; “We are all healthy until we get sick”. If active infection in these animals is documented by molecular testing or isolation, treatment is most likely indicated, especially if the infection can be eliminated and re-infection (through repeated tick or flea exposure) can be prevented.

Cross reactivity among various infectious agents is another limitation to serological testing. Cross reactivity among members of a genus should be an anticipated occurrence, such as the strong cross reaction between Ehrlichia canis and Ehrlichia chaffeensis or between Anaplasma phagocytophilum and Anaplasma platys. In these two examples, cross reactivity is of less clinical relevance, as in each instances both organisms with the genus respond to the same antibiotic, doxycycline. However cross reactivity between Babeisa canis and Babesia gibsoni is of clinical relevance as different drugs are used to eliminate these infections. Also, for an increasing number of infections (babesiosis, bartonellosis, leishmaniasis and others) the serological response to the organism may be minimal or non-existent, despite chronic, active infection with the organism. It is important to state that all diagnostic tests have limitations that can influence the diagnostic interpretation and how a patient is treated. When ever possible isolation, serological and molecular-based diagnostic testing should be performed in tandem. The notion that a clinician should pick a single best test or target only the most likely infectious agent in a given patient is naïve and not realistic in the clinical setting. Many infectious agents induce similar disease manifestations and it is increasingly obvious that polymicrobial infections exist more frequently than previously recognized. When confronted with a non-infectious disease process, such as cancer, thousands of dollars are spent on imaging and staging prior to the initiation of treatment. When an infectious disease is high on the list of differential diagnoses, testing dollars should be spent on determining the infectious agent or agents responsible for the disease process. Although molecular diagnostic tests have many inherent benefits, sensitivity is always an issue. Due to low template number in the test sample, a negative PCR result can never confirm that an animal is not infected with a specific pathogen. This is one of the most compelling arguments for combining serological and molecular testing modalities. NOVEL APPROACHES TO CULTURING BACTERIA In recent years, our research group has developed a novel approach to the isolation of fastidious bacteria. These efforts evolved out of our desire to isolate Bartonella species from dogs and people, as current microbiological approaches lack sensitivity. As a result of our development efforts, we found a liquid growth media, used to support the growth of insect cell lines, that will grow bacteria from patient samples where conventional approaches fail to achieve an isolate. Using this approach, we were able to isolate Mycobacterium kanssii from the pleural fluid of a dog and various bacteria form “culture-negative” pericardial fluids from racehorses residing in Kentucky. The clinical utility of this isolation approach is yet to be established, but preliminary results are most encouraging. Enhanced isolation techniques in conjunction with sensitive molecular detection techniques could drastically change perceptions related to the role of infectious agents in the expression of complex diseases, particularly autoimmune or idiopathic immune-mediated diseases. In the context of clinical medicine, it is possible that too much microbiological emphasis and excessive clinical relevance has been accorded organisms that are easy to isolate (Staphylococcus and Streptococcus species, Escherichia coli) from patient samples and not enough emphasis has been placed on organisms that are highly fastidious. In the future, the use of molecular based tests that can simultaneously detect multiple organisms will clarify complex interactions that influence pathogenesis, disease expression and treatment outcomes. This is truly an exciting time for infectious disease researchers and clinicians.

Selected References: 1. Birkenheuer AJ, Levy MG, Breitschwerdt EB. Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (asian genotype) and B. canis DNA in canine blood samples. J Clin Microbiol 41:4172-4177, 2003. 2. Kordick SK, Breitschwerdt EB, Hegarty BC, Southwick KL, Colitz CM, Hancock SI, Bradley JM, Rumbough R, Mcpherson JT, MacCormack JN. Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina. J Clin Microbiol 37:2631-2638, 1999. 3. Breitschwerdt EB, Hegarty BC, Hancock SI. Sequential evaluation of dogs naturally infected with Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or Bartonella vinsonii. J Clin Micrbiol 36:2645-2651, 1998. 4. Maggi RG, Chomel B, Hegarty BC, Henn J, Breitschwerdt EB. A Bartonella vinsonii berkhoffii typing scheme based upon 16S-23S ITS and Pap31 sequences from dog, coyote, gray fox, and human isolates. Mol Cell Probes 20:128-34, 2006. 5. Duncan AW, Maggi RG, Breitschwerdt EB. A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples: Pre-enrichment liquid culture followed by PCR and subculture onto agar plates. J Microbiol Methods 69:273-281, 2007. 6. Breitschwerdt EB, Maggi RG, Duncan AW, Nicholson WL, Hegarty BC, Woods CW. Bartonella species in blood of immunocompetent persons with animal and arthropod contact. Emerg Infect Dis 13:938-941, 2007. 7. PPVP Diniz, Maggi RG, Schwartz DS, Cadenas MB, Bradley JM, Hegarty BC, Breitschwerdt EB. Canine bartonellosis: Serological and molecular prevalence in Brazil and evidence of co-infection with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii. Vet Res 38:697-710, 2007. 8. Duncan AW, Maggi RG, Breitschwerdt EB. Bartonella DNA detected in saliva collected from dogs. Emerg Infect Dis.13:1948-1950, 2007. 9. Duncan AW, Marr HS, Birkenheuer AJ, Maggi RG, Williams LE, Correa MT, Breitschwerdt EB. Bartonella DNA in the blood and lymph nodes of Golden Retrievers with lymphoma and in healthy controls. J Vet Intern Med. 22:88-98; 2008. 10. Maggi RG, Breitschwerdt EB. Potential limitations of the 16S-23S rRNA intergenic region for the molecular detection of Bartonella species. J Clin Microbiol 43:1171-76, 2005. 11. Maggi RG, Compton SM, Trull CL, Mascarelli PE, Mozayeni BR, Breitschwerdt EB. Infection with hemotropic Mycoplasma sp. in people with and without extensive arthropod and animal contact. J Clin Microbiol 51:3237-3241, 2013. 12. Pultorak EL, Maggi RG, Mascarelli PE, Breitschwerdt EB. Serial testing from a three-day collection period using the BAPGM platform may enhance the sensitivity of Bartonella spp. detection in bacteremic human patients. J Clin Microbiol 57:1673-1677, 2013. 13. Hegarty BC, Bradley JM, Lappin MR, Balakrishnan N, Mascarelli PE, Breitschwerdt EB. Analysis of Seroreactivity against Cell Culture-derived Bartonella spp. antigens in dogs. J Vet Intern Med 28:38-41, 2014. 14. Breitschwerdt EB, Hegarty BC, BA, Qurollo BA, Saito TB, Maggi RG, Blanton LS, Bouyer BH. Intravascular persistence of Anaplasma platys, Ehrlichia chaffeensis, and Ehrlichia ewingii DNA in the blood of a dog and two family members. Parasit and Vect.7:298, 2014.

15. Balakrishnan N, Musulin S, Varanat M, Bradley JM, Breitschwerdt EB. Serological and molecular prevalence of selected canine vector borne pathogens in blood donor candidates, clinically healthy volunteers, and stray dogs in North Carolina. Parasit Vectors. 7: e116, 2014.

SEROLOGICAL AND MOLECULAR DIAGNOSIS OF INFECTIOUS DISEASES: Something old and something new. Part II

Edward B. Breitschwerdt, DVM, Diplomate ACVIM (Companion Animal Internal Medicine)

Center for Comparative Medicine and Translational Research Department of Clinical Sciences, College of Veterinary Medicine,

North Carolina State University, Raleigh, NC 27606

One advantage of experience (in this case, a 30 year career as an academic internist) is the eventual realization that our collective efforts to manage illness in our patients have numerous limitations. There are obvious practical limitations, such as time, money, equipment and expertise (or the lack thereof), that confront clinicians and pathologists on a daily basis. However, there are somewhat less obvious limitations that relate to the sensitivity and specificity of diagnostic testing and the failure or inability to pursue unusual or unknown pathogens in our patients. Molecular diagnostic approaches have begun to facilitate a “modern day” revolution in our understanding of the interactions of multiple infectious agents, the complexity of disease expression induced by acute or chronic infection, and the redefinition of “previously understood” diseases such as babesiosis and leishmaniasis. Although there is an increasing appreciation for the importance of co-infection or polymicrobial infection in animal and human diseases, there are considerable gaps in our current understanding of the interactions between viruses, bacteria, protozoa and fungi as interactive contributors to complex disease expression. BENEFITS OF MOLECULAR DIAGNOSTIC TESTING Without question various molecular based tools have facilitated tremendous advances in diagnosis of infectious diseases. PCR amplification of organism-specific DNA sequences can be accomplished in a matter of hours and in most instances the detection of a PCR amplicon confirms active infection with a specific organism. This approach has distinct advantages over culture, which can require incubation times ranging from days, to weeks, to months depending on the organism. In most instances, approaches that provide rapid culture results select for only a few more easily grown organisms. Although serology or examination of the cell mediated immune response will remain an important component of infectious disease diagnosis, these tests identify evidence of immune recognition of a pathogen. Failure to confirm active infection, cross reactivity among various infectious agents and for an increasing number of infections (babesiosis, bartonellosis, leishmaniasis and others) the serological response to the organism may be minimal or non-existent, despite chronic, active infection are limitations to the interpretation of serological test results in a given patient. In some instances, the advent of PCR and DNA sequencing has allowed for the detection, characterization and at times reclassification of previously unknown or uncultured organisms.1 A few examples include: 1. The contemporary discovery, rediscovery and reclassification of Bartonella species. Historically, two

members of this genus was classified as Rochilamea (Bartonella bacilliformis) and Rickettsia (Rickettsia quintana and subsequently Rochilamea quintana) species respectively. Although the genus Rochilamea is extinct, a recently discovered Bartonella species has been named Bartonella rochilamea in honor of the historical contributions of Henrique da Rocha-Lima. In 1992, only two Bartonella species (B. bacilliformis, B. quintana) were known to exist, whereas now there are greater than 20 Bartonella species or subspecies.

2. The reclassification of the Hemobartonella and Eperythrozoon in the genera Mycoplasma. As with Rochilamea, the Hemobartonella and Eperythrozoon genera no longer exist, as these organisms were reclassified as Mycoplasma species on the basis of 16S rDNA sequence similarity. As hemotropic Mycoplasma species have not yet been cultured, epidemiological and diagnostic studies were limited to visualization of the organism. PCR amplification and sequencing have facilitated a molecular-based reclassification of these organisms. In addition, PCR targeting specific Mycoplasma gene sequences has allowed for the more sensitive detection of these organisms in the blood of healthy and sick cats and dogs. As a result, depending on the population studied, a large percentage of healthy or sick cats can harbor M. hemofelis, Candidatus Mycoplasma haemominutum or Candidatus Mycoplasma turicensis.

3. The reclassification of Ehrlichia equi as Anaplasma phyagocytophilum. In the United States, E. equi was discovered and initially defined by clinicians and researchers at the University of California at Davis as an acute febrile illness of horses that is accompanied by edema and a mild degree of thrombocytopenia.

Progressively over the next 20 years, E. equi was found to induce an acute febrile illness, accompanied by thrombocytopenia in cats, dogs and human beings. Based upon 16S rRNA gene sequences derived from Anaplasma, Ehrlichia and Cowdria species E. equi was reclassified as an Anaplasma species. Subsequently, it has been recognized that various A. phagocytophilum strains induce disease in cats, dogs, horses and people throughout the northern hemisphere.

4. The reclassification of Cowdria ruminantium as Ehrlichia ruminantium. Historically, C. ruminantium, the causative agent of heartwater disease in cattle, has been a economically devastating tickborne infectious disease throughout much of Africa and some Caribbean Islands where Amblyomma variegatium ticks were imported on cattle shipped from Africa. Based upon the 16 rDNA sequence, C. ruminantium was reclassified as an Ehrlichia species. With the advent of PCR and DNA sequencing, evidence of E. ruminantium was reported in dogs and HIV infected people from South Africa. Although somewhat scientifically astounding, this observation may make phylogenetic and biological sense as it is now known that several members of the genus Ehrlichia, including E. canis, E. ewingii and E. chaffeensis can infect both dogs and people following transmission by a vector tick. Therefore, the use of more highly sensitive molecular-based detection assays has resulted in a breakdown of the perceived “species barrier” for several Ehrlichia species.

In some instances, the advent of PCR and DNA sequencing has allowed for the initial discovery (or rediscovery) of an entire genus of intravascular bacteria. A specific example includes the genus Bartonella: 1. In the United States Bartonella species were discovered in the early 1990s in association with efforts to

characterize “unculturable,” silver-staining bacteria observed in HIV-infected individuals with bacillary angiomatosis and peliosis hepatis. Using 16S rRNA eubacterial primers and DNA sequencing, Dr. David Relman at Stanford University amplified sequences from BA tissues that were most similar to R. quintana and a previously unreported DNA sequence, which proved to be Bartonella henselae. Subsequent research confirmed that all cases of vasoproliferative peliosis hepatitis are associated with B. henselae infection, whereas bacillary angiomatosis has been associated with the isolation or molecular detection of B. quintana, B. henselae and most recently B. bovis.

2. After B. henselae was successful isolated from the blood of an HIV infected individual (B. henselae Houston I, ATCC type strain) it was determined that cats were the primary reservoir hosts from which this organism was transmitted to humans. Infection in an immunosuppressed human population facilitated the initial recognition and molecular characterization of Bartonella species infection in the United States. Although the subsequent discoveries in the area of Bartonella research are too numerous to mention, B. quintana, historically associated with wars, famines and deprivation was found to be present in the U.S. for the first time in history. Our research group has subsequently described B. quintana infection in a cynomolgus monkey (Macaca fascicularis), in dogs with B. quintana endocarditis and in cats that putatively transmitted B. quintana to a woman by a bite.2 Bartonella henselae, as opposed to Afipia felis, is now considered the primary if not the sole cause of human cat scratch disease. Detection of Bartonella species infections in human beings and cats lead to the isolation of a novel Bartonella subspecies (B. vinsonii subsp. berkhoffii) from a dog with endocarditis.

3. During the past decade, an expanding number of Bartonella species (at least 22 species and subspecies) have been discovered in domestic and wild animals, which serve as the primary bacterial reservoirs from which Bartonella spp. are transmitted to human beings or to other non-host adapted animals via bites, scratches or following inoculation by a spectrum of arthropods (lice, fleas, sandflies, biting flies and potentially ticks). Cats, cows, dogs, mice, rabbits, rats and squirrels can experience chronic (months to years) blood-borne infections with host-adapted Bartonella species, with the potential for transmission by bites or scratches to people.

4. Our research group has emphasized studies that have resulted in the enhanced detection of Bartonella species in the blood of animals and humans using an optimized combined pre-enrichment culture medium and a highly sensitive real-time PCR.3 Pre-enrichment culture followed by PCR amplification of bacterial specific genes has resulted in substantial improvement in the ability to detect Bartonella spp. in the blood of people or animals that have substantial arthropod exposure or occupational animal contact.4 By targeting multiple genes, we have also documented that dogs and people can be infected with more than one Bartonella species.4,5 In addition, sequencing of specific genes or spacer regions can be used to genotype B. vinsonii subsp. berkhoffii which has epidemiologic and diagnostic importance.6

5. Bartonella species are now recognized as a cause of serious disease manifestations including arthritis, endocarditis, encephalitis, meningitis, hepatitis and lymphadenitis in dogs and people. It is very possible that these organisms represent an occupational risk for those individuals with extensive animal and arthropod exposure.

6. As most Bartonella species have been discovered in the past decade, there is still much to learn regarding routes of transmission and disease causing potential. Recently, we have documented a high prevalence of Bartonella infection in the lymph nodes of healthy Golden retrievers and Golden retrievers with lymphoma.7 In this same study, we were unable to detect Anaplasma or Ehrlichia DNA in the blood or lymph nodes of the study population.

LIMITATIONS OF MOLECULAR DIAGNOSTIC TESTING The basis of all molecular diagnostic testing is a cumulative genomic data base (Gen Bank), that provides DNA sequences, the source of these sequences and other information relative to deposit of the sequence into the data base. Unfortunately, there is substantial variation in the quality of he deposited sequence and the data that is provided relative to the source of the sequence. Although by definition a molecular based diagnostic test should be 100% specific based upon the premise that a unique gene sequence is being targeted, this is not always true or feasible for technical reasons. A few specific examples:

1. The 16S-23S intergenic spacer region has proven to be a valuable target for the molecular-based

diagnosis of Bartonella species infection. For the genus Bartonella, there is in most instances substantial variation among species within the ITS region. Therefore, using the ITS region as a molecular target would allow for both detection and speciation of the infecting Bartonella species in a single PCR reaction. When PCR primers were selected for this purpose by our laboratory and others, there were no Mesorhizobium (a plant adapted bacteria that is phylogenetically related to the genus Bartonella), ITS sequences deposited in Gen Bank. As would be expected, this plant bacteria is frequently found in water and unfortunately can contaminate molecular grade water resulting in a false positive diagnostic test results (amplification of Mesorhizobium DNA that is of the same amplicon size as a Bartonella species).8 In both our diagnostic (Vector Borne Diseases Diagnostic Laboratory) and research laboratories (Intracellular Pathogens Research Laboratory) we have a saying: A band on a gel is only a band on a gel until a DNA sequence from that specific band is available for confirmation. The advent of real-time (or quantitative PCR) with the use of melt curves has improved, but has not eliminated the potential for cross priming and misinterpretation of a PCR result.

2. Due to the available sequences and the relative high degree of genomic conservation among these species, most diagnostic laboratories target the 16S rRNA gene by PCR for the diagnosis of Anaplasma or Ehrlichia infection. In our laboratory a single PCR reaction is used to document infection with either species, if a positive result is obtained, then 5 independent PCR reactions are used to determine if the sample is infected with E. canis, E. chaffeensis, E. ewingii, A. phagocytophilum or A. platys. This allows us to determine the infecting species, which has clinical, epidemiological and zoonotic implications and allows for the detection of co-infection with more than one Anaplasma and Ehrlichia species.9 Post-treatment PCR testing can also be used to support therapeutic elimination of infection with an Ehrlichia species.10 Unfortunately, all bacteria are believed to have one or more 16S rRNA genes, most of which have been conserved in for millions of years. When a diagnostic test targets a gene that is highly conserved among numerous bacterial species, cross priming can result in multiple bands or bands of an appropriate size for the targeted organism because of contamination of molecular grade water, the Taq polymerase or the primers with bacterial DNA. Although a potentially valuable diagnostic tool, assays that non-specifically target the 16S rRNA gene (i.e. eubacterial primers) can be technically problematic for a number of reasons.

CAUTIONS REGARDING MOLECULAR DIAGNOSTIC TESTING The international genome data base (Gen Bank) is available to anyone with computer access. Therefore the sequences required for the design of a molecular diagnostic test are also available to anyone. Kits can be purchased for DNA extraction and thermocyclers for PCR amplification are no longer cost prohibitive for many commercial laboratories. Unfortunately, technical expertise and rigid quality control is absolutely critical for the accurate performance of molecular diagnostic testing. Many practicing veterinarians are not familiar with the strengths and limitations of this diagnostic approach, therefore assistance in the interpretation of both positive and negative test results is frequently required. There is no standardization or

quality control testing among laboratories providing molecular diagnostic test results to the public. As is stated for other consumables, “Let the buyer beware!” There are numerous other issues that complicate and challenge the current state of the art in molecular diagnostic testing. A few of these include: 1. An exceptional well designed PCR assay that will reproducibly detect a low genome copy number (1-2

copies of an organism-specific gene target in a 200ul sample extraction) may not work as efficiently or may not work at all if transferred to a new (same manufacturer) or different thermocycler (different laboratory or different thermocycler manufacturer).

2. PCR contamination is a constant fear for the Director of a molecular diagnostic laboratory. Unfortunately, despite appropriate controls, it is impossible to prove that a given PCR amplicon obtained from a patient sample is not a function of PCR contamination in the laboratory. Although a serious concern, the use of negative controls (extraction control, PCR control) in conjunction with laboratory surveillance greatly minimizes this concern. For technical reasons, the advent of real-time PCR has further minimized this concern.

3. With the use of samples such as paraffin blocks, specific protocols must be followed to avoid DNA carry over during the collection and extraction process, which has resulted in the reporting of false positive test results and data misinterpretation in the literature.

MOLECULAR DIAGNSOTICS, PCR TESTING: DO’S AND DON’TS:

PCR = Polymerase Chain Reaction

PCR is a technique used to amplify DNA or RNA (Reverse Transcriptase PCR) PCR is a highly sensitive diagnostic technique that can be used to detect small quantities of bacterial, viral or protozoal DNA in patient blood, fluid or tissue specimens. PCR does not amplify or detect antibodies or antigens, only DNA or RNA. Therefore the targeted organism must be in the sample for DNA or RNA to be detected. WHEN TO USE PCR TESTING: 1. Use PCR prior to administration of an antibiotic or antiprotozoal drug to confirm active infection (i.e.

presence of DNA equals presence of the organism). Antibody tests confirm exposure to the organism and may or may not be reflective of active infection. When in doubt, store an EDTA-anti-coagulated blood sample in the refrigerator prior to administering treatments. “It is better to have a pre-treatment sample and not need it, than to need the sample and not have it”. Avoid formalin!

2. Use PCR following completion of treatment to confirm therapeutic elimination of the infection (i.e. failure to detect DNA supports treatment success). Conceptually veterinarians can think of PCR testing using the same principles associated with culturing urine. It is best to perform PCR prior to antibiotic administration or at some time point following treatment. If treatment has not eliminated the infection, waiting 2-3 weeks following treatment should allow the organism to increase in the blood to a level that can be detected by PCR.

3. Use PCR testing when the species of an infectious agent is important for determination of the appropriate type of drug to use for treatment. For example, different drugs would be used to treat Babesia canis and Babesia gibsoni infections in dogs. Species-specific PCR allows us to differentiate the infecting species.

PCR: Points to Ponder: 1. Although very sensitive tests, a negative PCR result will never definitively eliminate the possibility of an

infectious agent. 2. Repeated negative PCR results would strongly support therapeutic elimination of the infectious agent. 3. The use of glucocorticoids will in most instances increase the number of infectious particles in the blood.

Therefore corticosteroid administration, particularly at immunosuppressive doses, can enhance PCR detection of an infectious agent.

4. If the PCR test is properly designed and properly performed, a false positive result should not occur. PCR contamination (i.e. in laboratory contamination with PCR products) can result in a false positive result. However, molecular diagnostic laboratories run controls to help avoid or to detect PCR contamination. Newer PCR approaches such as real-time PCR greatly decrease the possibility of PCR contamination.

5. PCR assays performed by different laboratories can vary substantially in quality. Always know your laboratory.

Selected References: 1. Birkenheuer AJ, Levy MG, Breitschwerdt EB. Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (asian genotype) and B. canis DNA in canine blood samples. J Clin Microbiol 41:4172-4177, 2003. 2. Kordick SK, Breitschwerdt EB, Hegarty BC, Southwick KL, Colitz CM, Hancock SI, Bradley JM, Rumbough R, Mcpherson JT, MacCormack JN. Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina. J Clin Microbiol 37:2631-2638, 1999. 3. Breitschwerdt EB, Hegarty BC, Hancock SI. Sequential evaluation of dogs naturally infected with Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or Bartonella vinsonii. J Clin Micrbiol 36:2645-2651, 1998. 4. Maggi RG, Chomel B, Hegarty BC, Henn J, Breitschwerdt EB. A Bartonella vinsonii berkhoffii typing scheme based upon 16S-23S ITS and Pap31 sequences from dog, coyote, gray fox, and human isolates. Mol Cell Probes 20:128-34, 2006. 5. Duncan AW, Maggi RG, Breitschwerdt EB. A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples: Pre-enrichment liquid culture followed by PCR and subculture onto agar plates. J Microbiol Methods 69:273-281, 2007. 6. Breitschwerdt EB, Maggi RG, Duncan AW, Nicholson WL, Hegarty BC, Woods CW. Bartonella species in blood of immunocompetent persons with animal and arthropod contact. Emerg Infect Dis 13:938-941, 2007. 7. PPVP Diniz, Maggi RG, Schwartz DS, Cadenas MB, Bradley JM, Hegarty BC, Breitschwerdt EB. Canine bartonellosis: Serological and molecular prevalence in Brazil and evidence of co-infection with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii. Vet Res 38:697-710, 2007. 8. Duncan AW, Maggi RG, Breitschwerdt EB. Bartonella DNA detected in saliva collected from dogs. Emerg Infect Dis.13:1948-1950, 2007. 9. Duncan AW, Marr HS, Birkenheuer AJ, Maggi RG, Williams LE, Correa MT, Breitschwerdt EB. Bartonella DNA in the blood and lymph nodes of Golden Retrievers with lymphoma and in healthy controls. J Vet Intern Med. 22:88-98; 2008. 10. Maggi RG, Breitschwerdt EB. Potential limitations of the 16S-23S rRNA intergenic region for the molecular detection of Bartonella species. J Clin Microbiol 43:1171-76, 2005. 11. Maggi RG, Compton SM, Trull CL, Mascarelli PE, Mozayeni BR, Breitschwerdt EB. Infection with hemotropic Mycoplasma sp. in people with and without extensive arthropod and animal contact. J Clin Microbiol 51:3237-3241, 2013. 12. Pultorak EL, Maggi RG, Mascarelli PE, Breitschwerdt EB. Serial testing from a three-day collection period using the BAPGM platform may enhance the sensitivity of Bartonella spp. detection in bacteremic human patients. J Clin Microbiol 57:1673-1677, 2013. 13. Hegarty BC, Bradley JM, Lappin MR, Balakrishnan N, Mascarelli PE, Breitschwerdt EB. Analysis of Seroreactivity against Cell Culture-derived Bartonella spp. antigens in dogs. J Vet Intern Med 28:38-41, 2014. 14. Breitschwerdt EB, Hegarty BC, BA, Qurollo BA, Saito TB, Maggi RG, Blanton LS, Bouyer BH. Intravascular persistence of Anaplasma platys, Ehrlichia chaffeensis, and Ehrlichia ewingii DNA in the blood of a dog and two family members. Parasit and Vect.7:298, 2014. 15. Balakrishnan N, Musulin S, Varanat M, Bradley JM, Breitschwerdt EB. Serological and molecular prevalence of selected canine vector borne pathogens in blood donor candidates, clinically healthy volunteers, and stray dogs in North Carolina. Parasit Vectors. 7: e116, 2014.

CAT SCRATCH DISEASE AND FELINE BARTONELLOSIS

Edward B. Breitschwerdt DVM, Diplomate ACVIM Comparative Medicine Institute

Department of Clinical Sciences, College of Veterinary Medicine North Carolina State University, Raleigh, NC 27606

And Galaxy Diagnostics Inc.

Research Triangle Park, NC 27709

The genus Bartonella is currently comprised of at least 30 species and subspecies of vector-transmitted, fastidious, gram-negative bacteria that are highly adapted to one or more mammalian reservoir hosts. On an evolutionary basis, Bartonella henselae, Bartonella clarridgeae and Bartonella koehlerae, and potentially Bartonella quintana have evolved to cause persistent intravascular infection in domestic cats and wild felid species. In contrast, Bartonella vinsonii subspecies berkhoffii has evolved to cause persistent intravascular infection in dogs and wild canines, including coyotes and foxes, but was recently isolated from a young cat with recurrent ostomyelitis. Other Bartonella species have evolved to cause persistent blood borne infection in rodents, small mammals or ruminants, for example Bartonella bovis. Epidemiological evidence and experimental flea transmission studies support an important role for fleas in the transmission of B. henselae, B. clarridgeae and most likely B. koehlerae among cats. Two other Bartonella species, B. bovis and B. quintana have been isolated from cat blood, but the modes of transmission and the reservoir potential of these species in felids has not been definitively established. Recently, we isolated Bartonella vinsonii subsp. berkhoffii from a cat with recurrent osteomyelitis. Although there is clinical and epidemiological evidence to support tick transmission of B. vinsonii subspecies berkhoffii to dogs and coyotes, the mode of transmission of this Bartonella species to cats and dogs has not been determined. Recently, Bartonella vinsonii subsp. berkhoffii DNA was amplified from fleas collected from dogs in Florida, suggesting that fleas could also play a role in the transmission of this subspecies. As reviewed in several publications, numerous domestic and wild animals, including bovine, canine, feline, human, and rodent species can serve as chronically infected reservoir hosts, thereby supporting zoonotic transmission for various Bartonella species. In addition to the large number of documented reservoir hosts, an increasing number of arthropod vectors, including biting flies, fleas, keds, lice, sandflys and ticks have been implicated in the transmission of Bartonella species. Considering the diversity of Bartonella species and subspecies, the large number of reservoir hosts and the spectrum of arthropod vectors, the clinical and diagnostic challenges posed by Bartonella transmission in nature appear to be much more complex than is currently appreciated in human and veterinary medicine.

Once an animal is infected by a bite, a scratch or arthropod, Bartonella species localize

to erythrocytes and endothelial cells, which provides a potentially unique strategy for bacterial persistence within the blood stream of reservoir or non-reservoir species. In vitro infection of human CD34+ progenitor cells with B. henselae suggests that these bacteria are capable of infecting bone marrow progenitor cells which may contribute to ongoing erythrocytic infection.

Infection of bone marrow progenitor cells followed by non-hemolytic intracellular colonization of erythrocytes would preserve Bartonella organisms for efficient vector transmission, protect Bartonella from the host immune response, facilitate widespread vascular dispersion throughout the tissues of the body, and potentially contribute to decreased antimicrobial efficacy. As Bartonella spp. represent the only genus of bacteria known to infect bone marrow progenitor cells, this bacteria could play a role in the pathogenesis of various bone marrow diseases.

Feline Bartonellosis The extent to which members of the genus Bartonella are pathogenic for cats remains to be determined. Due to flea exposure, B. henselae bacteremia can be documented in 25 to 41% of healthy cats in different regions throughout the world. Self-limiting febrile illness of 48 to 72 hours

duration, mild to moderate transient anemia, and transient neurologic dysfunction was reported in cats experimentally infected with B. henselae by blood transfusion. Self-limiting fever can also occur in B. henselae bacteremic cats following minor surgical procedures. Although unproven, it is likely that stress, such as surgery or trauma, can induce transient disease manifestations in cats, including self-limiting fever, mild anemia and neurological dysfunction. Due to the high percentage of chronically bacteremic healthy cats in the United States, establishing a cause and effect relationship between disease manifestations and bacteremia in cats has required large epidemiological studies in Bartonella endemic and non-edemic regions. Seroepidemiologic studies have generated contrasting results, as to whether fever, lymphadenopathy, stomatitis and gingivitis are caused by B. henselae. Bartonella henselae DNA and intrathecal antibody production has also been demonstrated in cats with neurological disease. Recent evidence documents variation in the virulence of various B. henselae strains. More highly pathogenic strains can induce myocarditis and sudden death in young cats or kittens. Also, iImmunosupression associated with FeLV or FIV appears to increase the pathogenicity of B. henselae infection in cats. In experimentally infected cats, fever, lymphadenopathy, mild neurological signs and reproductive disorders have been reported. In experimentally-infected cats, gross necropsy results are unremarkable; however, histopathological lesions can include peripheral lymph node hyperplasia, splenic follicular hyperplasia, lymphocytic cholangitis/pericholangitis, lymphocytic hepatitis, lymphoplasmacytic myocarditis, and interstitial lymphocytic nephritis. These findings would indicate that antibiotic treatment should be considered in seroreactive or bacteremic cats with these disease manifestations.

The diagnosis of Bartonella infection should be confirmed by culturing the organism from blood or tissues such as lymph node or heart valve (endocarditis) or by amplifying Bartonella-specific DNA sequences from blood or tissues using PCR. Bartonella henselae, and B. quintana can be visualized within (not on the surface of) erythrocytes using confocal or electron microscopy. Cell lysis, using a commercially available lysis centrifugation technique or by freezing the blood sample prior to plating, facilitates bacterial isolation of B. henselae from blood, but is most likely less sensitive for the isolation of other Bartonella spp. Although organisms within the genus Bartonella are fastidious and slow-growing, B. henselae and B. clarridgeae can be cultured successfully with agar plates containing 5% defibrinated rabbit or sheep blood, that are maintained at 35oC in a high humidity chamber with a 5% CO2 concentration. In our experience, bacterial colonies may not be visible until 10 to 56 days after inoculation of the agar plate. As cats maintain a higher level of bacteremia, culturing B. henselae or B. clarridgeiae from aseptically obtained blood samples is much more likely to be successful than culturing B. henselae from a dog or human blood sample. The recent introduction of a liquid insect cell culture-based growth medium (Bartonella alpha Proteobacteria growth medium) has facilitated the successful isolation of B. henselae from dog and human blood samples. Also the use of BAPGM allowed us to isolate B. vinsonii subsp. berkhoffii from the cat with osteomyelitis. In conjunction with IFA serology, the BAPGM diagnostic platform is available from Galaxy Diagnostics (www.galaxydx.com) for testing cats with suspected bartonellosis.

Because members of the order Rickettsiales are cultivable only in host cells or viable tissue culture cells, cultivation of Bartonella species on bacteriologic media, in conjunction with DNA divergence studies, provided the justification for removal of members of the genus Bartonella from the order Rickettsiales. In tissues, Bartonella are small, curved, gram-negative rods that stain positively with silver stains such as the Warthin-Starry stain, although this approach is diagnostically insensitive in most instances. PCR assays targeting several Bartonella-specific gene sequences have been described and should be used to confirm infection with a Bartonella species infection in tissues or blood.

Seroconversion, detected by IFA or ELISA techniques, can be documented in people with acute disease and in cats and dogs following experimental infection. The kinetics of the serologic response to B. henselae antigens in chronically-infected experimental cats is highly variable in degree and duration. A seroepidemiologic survey, incorporating 577 samples from throughout North America identified an overall prevalence of 28%, with prevalence rates ranging from a low of 4–7% in the Midwest and Great Plains region to 60% in the southeast. High seroprevalence rates correlate

with warm, humid climates conducive for the environmental maintenance of cat fleas, which have been shown to be capable of transmitting B. henselae from cat to cat. As mentioned above, it is likely that most cats are bacteremic with the less virulent strains of B. henselae. Virulence characteristics are poorly defined for the other Bartonella sp. that infect cats. A seroepidemiologic study of 592 cats in Baltimore, MD, identified an overall seroprevalence rate of 14.7%, with a much higher prevalence in feral cats (44.4%) as compared to pet cats donated to the City Municipal Animal Shelter (12.2%). Although providing useful epidemiologic data, serologic test results may be of limited clinical utility for several reasons. We have been unable to detect B. henselae-specific antibodies in some bacteremic cats, whereas in other cats we have been unable to culture Bartonella when antibodies are detectable. In recent prospective feline clinical studies, the detection of antibodies was not predictive for the detection of DNA, which is a strong indication of active infection. Negative blood cultures obtained from cats seroreactive to B. henselae antigens may reflect low level bacteremia or the timing of the blood culture, since experimentally-infected cats experience a relapsing pattern of bacteremia. Numerous naturally-infected cats are bacteremic for months to years, generally in conjunction with low, negative or non-diagnostic antibody titers. In our experience, high antibody titers generally correlate with positive blood cultures or detection of Bartonella DNA in the blood using the BAPGM enrichment blood culture (or other fluid sample culture) platform. The extent of serologic cross reactivity among Bartonella species requires additional clarification, however, both dogs and human patients develop a species-specific serological response during the early stage of infection. We and others have demonstrated co-infection with B. henselae, B. clarridgeiae, B. bovis or B. quintana in cats, thus seroreactivity to more than one Bartonella sp. antigen may reflect prior exposure to (and potentially active infection with) multiple species within the genus.

Because of disparate results among studies and an overall lack of microbiologic data in clinical therapeutic trails, numerous issues related to treatment of Bartonella infection remain controversial. In contrast to the apparent lack of response to antimicrobial treatment in human CSD patients, bacillary angiomatosis, parenchymal bacillary peliosis, and acute Bartonella bacteremia appears to respond to antimicrobial treatment, even in immunocompromised (predominantly HIV-infected individuals). In humans, doxycycline, erythromycin and rifampin are recommended antibiotics, but clinical improvement has been reported following the use of penicillin, gentamicin, ceftriaxone, ciprofloxacin, and azithromycin. Treatment for 2 weeks in immunocompetent individuals and 6 weeks in immunocompromised people is generally recommended. Relapses, associated with bacteremia, have been reported in immunocompromised people despite treatment for 6 weeks. Antimicrobial efficacy has not been established for any antibiotic for eliminating B. henselae bacteremia in cats. Results from our laboratory and others indicate incomplete treatment responses in cats treated for 2 or 4 weeks with doxycycline or enrofloxacin. Cats experimentally-infected with B. henselae do not develop protective immunity to a heterologous challenge with B. clarridgeiae or other B. henselae strains. Differences in results derived from cats infected with B. henselae by blood transfusion from an infected cat or by flea transmission are most likely due to alterations in virulence, induced during in vitro culture and subsequent experimental inoculation of cultured organisms. Thus experimental infection studies that incorporate vector transmission of a Bartonella sp. are recommended as these studies more likely generate data that is analogous to what occurs in nature.

Cat Scratch Disease

For over a century regional lymphadenopathy has been associated with animal contact, particularly cat scratches. Over the years, numerous microorganisms were implicated as the cause of CSD. In 1983, small, argyrophilic (easily impregnated with silver), gram-negative, pleomorphic bacteria were seen within blood vessel walls and macrophages in lymph nodes of patients with CSD. In 1988, a bacteria, later designated Afipia felis, was cultured from lymph nodes of CSD patients. In the same year, Cockerell and colleagues proposed a possible association between epithelioid angiomatosis and CSD in a letter to Lancet. In 1992, Regnery and colleagues at the Centers for Disease Control, identified seroreactivity to B. henselae antigens in 88% of 41 patients with suspected CSD compared to 3% of controls. Similarly, a case-controlled Connecticut study of CSD patients and their cats identified a strong association with cats 12 months of age or younger, a history of a scratch or bite, contact with fleas, and seroreactivity to B. henselae antigen. Additional

support that B. henselae is the predominant cause of CSD was provided when Bartonella DNA was amplified from lymph node samples of 21 of 25 (84%) patients with suspected CSD, using a polymerase chain reaction assay. A similar study from Sweden identified B. henselae DNA, but failed to identify A. felis DNA, in a large number of patients with suspected CSD. Subsequently, we cultured Bartonella species from 17 of 19 cats owned by 14 patients with CSD, which indicates that bacteremia is a frequent occurrence in cats that transmit B. henselae to a human being. Studies to date indicate that B. henselae is the predominant, but not the sole cause of CSD. In 1995, Clarridge et al. isolated a novel Bartonella species, which was named B. clarridgeiae, from a cat belonging to a patient infected with HIV from whom B. henselae was isolated. Our studies have implicated B. clarridgeiae as a cause of inoculation papules, fever and regional lymphadenopathy (CSD) in 3 people, however to date, B. clarridgeiae DNA has not been detected in lymph nodes of CSD patients and the organism has not been isolated from blood or lymph nodes of these patients.

Historically, atypical manifestations of CSD have included tonsillitis, encephalitis, cerebral arthritis, transverse myelitis, granulomatous hepatitis and/or splenitis, osteolysis, pneumonia, pleural effusion, and thrombocytopenic purpura. With the advent of specific diagnostic techniques, (culture, serology, and PCR), there has been a dramatic increase in reports describing patients with atypical manifestations of CSD. Osteomyelitis, granulomatous hepatitis and granulomatous splenitis have been increasingly recognized in children infected with B. henselae, who frequently lack the classical lymphadenopathy of CSD. Previously, Bartonella infection would not have been considered a likely differential diagnosis by the physician in patients lacking a history of lymphadenopathy or animal contact. As evidenced by reports in the past four years, the spectrum of human disease associated with the genus Bartonella continues to expand, requiring periodic reassessment as new information becomes available.

Based upon recent advances in our knowledge of the zoonotic potential of members of the genus Bartonella, the designations cat scratch disease and cat scratch fever may be most appropriate when considering human disease manifestations from a historical perspective. Because cat scratch disease generally denotes a self-limiting illness characterized by fever and lymphadenopathy and because the recognized spectrum of human disease manifestations associated with Bartonella infections (which may not include fever or lymphadenopathy) has expanded considerably in recent years, it is becoming obvious that the designation CSD lacks clinical, microbiologic and zoonotic utility. Although cats are a major reservoir for B. henselae and potentially B. clarridgeiae, some patients deny the possibility of a cat scratch or bite wound, or indicate no contact with cats. Transmission from environmental sources, arthropod vectors or other animal hosts is probable and the more inclusive term bartonellosis may facilitate enhanced future understanding of diseases caused by members of the genus Bartonella. In this context, our laboratory has reported B. henselae infections in dogs and human patients after exposure to tropical rat mites and wood louse hunter spiders.

Although recent research findings have substantially improved our understanding of the clinical, microbiologic and zoonotic aspects of diseases caused by Bartonella species, the exact mode of transmission, the relative role of various insect vectors such as fleas and ticks, the identification of potential reservoir hosts, and the spectrum of animal and human illnesses caused by these organisms remains largely undetermined. For example, although it is well established that the human body louse transmits B. quintana, the reservoir and mode of transmission that results in bacillary angiomatosis in the United States has not been established. The pathogenic potential of these organisms appears to be of considerable importance in cats, dogs, as well as immunocompromised and immunocompetent people.

BARTONELLOSIS: A ONE HEALTH PERSPECTIVES ON AN EMERGING ZOONOTIC INFECTIOUS DISEASE

Edward B. Breitschwerdt, DVM, DACVIM, Comparative Medicine Institute,

College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA Bartonella species are fastidious Gram-negative bacteria that are highly adapted to a mammalian reservoir host and within which the bacteria usually cause a long-lasting intra-erythrocytic bacteremia.1-3 These facts are of particular importance to veterinarians and physicians, as an increasing number of animal reservoir hosts have been identified for various Bartonella species. Among numerous other examples, Bartonella henselae has co-evolved with cats, Bartonella vinsonii subsp. berkhoffii has co-evolved with dogs and wild canines, and Bartonella bovis has co-evolved with cattle. Importantly, the list of reservoir-adapted Bartonella species, including a large number of rodent species that might serve as “pocket pets”, continues to grow exponentially, as new Bartonella spp. are discovered.2-3 Prior to 1990, there were only two named Bartonella species, whereas there are now at least 36 named and numerous unnamed or candidatus species, based upon deposited Gen Bank sequences or preliminary reports, respectively. In the natural reservoir host, chronic bacteremia with a Bartonella species can frequently be detected by blood culture or PCR in outwardly healthy individuals. In contrast, the diagnostic detection of a Bartonella spp. in a non-reservoir adapted host can be extremely difficult. Most, although not all diseases caused by Bartonella spp. occur in accidental hosts and these organisms are being increasingly implicated as a cause of zoonotic infections.4-8 Until recently, mechanisms that facilitate persistent Bartonella bacteremia in mammals were not well understood. Recent reports have identified an intra-endothelial and intra-erythrocytic localization for these bacteria, which represents a unique strategy for bacterial persistence.2,3 Non-hemolytic intracellular colonization of erythrocytes and endothelial cells would preserve the organisms for efficient vector transmission, protect Bartonella from the host immune response, and potentially contribute to decreased antimicrobial efficacy. Other in vitro studies indicate that Bartonella spp. can infect dendritic cells, microglial cells, pericytes, monocytes and CD34+ bone marrow progenitor cells. Epidemiology Bartonella henselae was initially isolated from an HIV-infected human and subsequently from cats, dogs, horses, marine mammals and other small terrestrial mammals. Bartonella vinsonii subsp. berkhoffii was initially isolated from a dog with endocarditis in our North Carolina laboratory in 19939 and subsequently from cats, coyotes and human patients. Retrospectively, long-term administration of immunosuppressive doses of corticosteroids for a presumptive diagnosis of systemic lupus erythematosus with cutaneous vasculitis may have facilitated the isolation of the original type strain of B. vinsonii (berkhoffii) from this dog that subsequently developed endocarditis. Due to the relatively recent recognition that dogs can be infected with B. vinsonii (berkhoffii), B. henselae and potentially other Bartonella spp., seroprevalence data is somewhat limited.3 Seroprevalence was determined in 1,920 sick dogs from North Carolina or surrounding states that were evaluated at a veterinary teaching hospital. Using a reciprocal titer of >32, only 3.6% of sick dogs had antibodies to B. vinsonii (berkhoffii). Risk factors that could be associated with seroreactivity included: heavy tick exposure (Odds ratio 14.2), cattle exposure (OR 9.3), rural vs. urban environment (OR 7.1) and heavy flea exposure (OR 5.6). These data were interpreted to support the possibility that exposure to B. vinsonii (berkhoffii) was more likely in dogs in rural environments that were allowed to roam. In addition, these dogs were likely to have a history of heavy tick and flea infestations. Experimental flea transmission of B. henselae to dogs has now been confirmed in the laboratory (Lappin and Breitschwerdt, unpublished data). Also, cross reactivity to Bartonella antigens was not detected when testing sera from dogs experimentally infected with R. rickettsii or Ehrlichia canis. However, 36% of serum samples derived from dogs naturally infected with E. canis were reactive to B. vinsonii antigens. As E. canis is transmitted by Rhipicephalus sanguineous, this tick may be involved in the transmission of B. vinsonii. The possibility of tick transmission was further supported by two additional studies involving dogs infected with one or more Ehrlichia spp. from the same geographic region, in which seroreactivity to B. vinsonii (berkhoffii) antigens was 30% and 89%, respectively. Seroprevalence, using B. vinsonii (berkhoffii) antigens, was 10% (4/40 dogs) in dogs with suspected tick-borne illness from Israel and 36% in dogs with fever and thrombocytopenia from Thailand. Using an ELISA assay, 35% of 869 samples, derived from coyotes in California, contained antibodies to B. vinsonii (berkhoffii) antigens. Current data indicates that canine and human exposure to B. henselae and B. vinsonii (berkhoffii) can be found throughout much of the United States and most tropical and subtropical regions of the world.

Studies from Hawaii, the United Kingdom and Japan identified B. henselae seroprevalences of 6.5% (2/31dogs), 3.0% (3/100 dogs) and 7.7% (4/52). In our laboratory, B. henselae is the most common Bartonella species found in sick dogs using the BAPGM enrichment blood culture platform. The pathogenicity of all Bartonella spp. in dogs is poorly understood; however, like humans, dogs can be infected with numerous Bartonella spp. B. henselae DNA has been amplified and sequenced from the livers of dogs with peliosis hepatis, a unique pathological lesion also reported in B. henselae infected people. B. henselae DNA was also amplified from a dog and a horse with granulomatous hepatitis, a

histopathological lesion reported with some frequency in B. henselae-infected children and adults. Bartonella clarridgeae DNA has been amplified and sequenced from the liver of a Doberman pincher with copper storage disease and from the aortic valve of a dog with vegetative valvular endocarditis. Bartonella elizabethae, a species that infects rodents, was found in a dog that had experienced chronic weight loss culminating in sudden unexplained death. Based upon a large seroepidemiological, controlled study from the University of California (Davis), dogs that were seroreactive to either B. henselae, Bartonella clarridgeae or B. vinsonii (berkhoffii) were referred for evaluation of lameness, neutrophilic polyarthritis, nasal discharge, epistaxis and splenomegaly.10

In cats, B. henselae and B. clarridgeiae have been amplified or grown most frequently from cats and the fleas

collected from cats. Flea-associated transmission has been well documented amongst cats. In flea endemic areas, Bartonella spp. seroprevalence rates in cats can be greater than 90% and bacteremia rates can be greater than 50%. Granulomatous myocarditis has been reported in cats, naturally or experimentally-infected with B. henselae. B. vinsonii (berkhoffii) caused recurrent osteomyelitis in a cat.3,11 Pathogenesis Although as yet unproven, B. vinsonii (berkhoffii) may be transmitted to dogs by the bite of an infected flea or tick. Based upon antidotal evidence, dogs may also become infected with B. henselae by a cat bite or scratch, analogous to cat scratch disease in people. B. vinsonii appears to cause chronic intra-erythrocytic and endothelial cell infections dogs for extended periods of time, potentially resulting in vasoproliferative pathologies. Similar to other highly adapted intracellular vector-transmitted pathogens, the factors that ultimately result in these bacteria causing disease manifestations are likely multifactorial and as yet to be defined. If similar to babesiosis, another intra-erythrocytic pathogen, stress, hard work, parturition, concurrent infection with other organisms or therapeutic immunosuppression may contribute to the development of pathology. Following experimental inoculation of SPF dogs with culture grown B. vinsonii (berkhoffii), there was sustained suppression of peripheral blood CD8+ lymphocytes, accompanied by an altered cell surface phenotype and an increase in CD4+ lymphocytes in the peripheral lymph nodes.11 Therefore, infection with B. vinsonii (berkhoffii) appears to induce a degree of chronic immunosuppression that might predispose dogs to other infectious agents, resulting in a wide array of clinical manifestations in naturally-infected dogs. The pathogenesis of disease (myocarditis or endocarditis) in cats may ultimately be associated with the virulence of the specific strain. Cats infected with Bartonella spp. are commonly co-infected with hemoplasmas and at times, more than one Bartonella sp. However, whether co-infections magnify disease manifestations of either genera is unclear and in most studies co-infections did not appear to potentiate illness. From an evolutionary perspective, it is obvious that vectors, vector-borne organisms, and animal and human hosts have developed a highly adapted form of interaction. In general, vectors need blood for nutrition; bacterial, rickettsial and protozoal organisms need an intracellular environment to survive, and immunologically, most hosts appear to be able to support chronic infection with vector-borne organisms for months to years without obvious deleterious effects. These factors serve to illustrate the potential difficulty in establishing causation in cats, dogs or people infected with a single or co-infected with multiple tick-transmitted pathogens. Recently, we proposed an addition to Koch’s postulates entitled the Postulate of Comparative Infectious Disease Causation.11 By satisfying this postulate, Bartonella species appear to be able to cause, endocarditis, granulomatous inflammatory diseases, particularly involving heart, liver, lymph nodes, and spleen, persistent intravascular infections and the induction of vasoproliferative tumors in animals and human patients.3,9,12-14

Clinical findings The spectrum of disease associated with Bartonella infection in dogs and most other animal species is currently

unknown. Endocarditis, has been reported in cats, cows, dogs, humans, and wildlife infected with a spectrum of Bartonella spp. In some dogs, intermittent lameness, bone pain, epistasis or fever of unknown origin can precede the diagnosis of endocarditis for several months, whereas other dogs will present with an acute history of cardiopulmonary decompensation.3,9 Cardiac arrhythmias secondary to myocarditis can be detected in cats and dogs without echocardiographic evidence of endocarditis. Granulomatous lymphadenitis has been associated with B. vinsonii (berkhoffii) and B. henselae in dogs. B. vinsonii (berkhoffii) and other Bartonella species appear to contribute to the development of dermatologic lesions indicative of a cutaneous vasculitis, panniculitis, as well as anterior uveitis, polyarthritis, meningoencephalitis and immune-mediated hemolytic anemia.3,10-15 Additional research efforts, using carefully designed case controlled studies are necessary to establish the frequency and extent to which Bartonella spp. contribute to dermatological, ocular, orthopedic, neurological or hematological abnormalities in dogs (and humans).

Clinically, many disease manifestations have also been attributed to Bartonella spp. infections in cats.3 However,

it is very difficult to prove disease associations in cats in the field because of the high prevalence rates in non-clinical carriers. In research cats that are infected by exposure to C. felis, fever, endocarditis, and myocarditis are the most common disease manifestations. As discussed for dogs, additional case controlled studies are needed in cats.

Diagnosis

Thrombocytopenia, anemia, which frequently can be immune-mediated, and neutropenia or neutrophilic leukocytosis are the hematological abnormalities in dogs that are seroreactive or BAPGM enrichment blood culture/PCR

positive.12,15 Thrombocytopenia is found in approximately half, eosinophilia approximately one third of infected dogs and monocytosis frequently occurs in Bartonella endocarditis. Hematological abnormalities have been rarely reported in cats, but similar to dogs, a subset of Bartonella-infected cats are neutropenic. Serum biochemical abnormalities are usually very mild or nonexistent in both cats and dogs. In cats, Bartonella spp. antibodies have correlated with polyclonal hyperglobulinemia and hypoglycemia.16

As B. henselae, B. koehlerae and B. vinsonii (berkhoffii) antibodies are infrequently detected (<4%) in sick dogs in

North America, detection of Bartonella spp. antibodies in a sick dog provides diagnostic support for prior exposure and potentially active infection. For this reason, treatment of seroreactive dogs or dogs from which any Bartonella spp. DNA is detected in blood or tissue samples would be recommended. Isolation and Molecular Detection of Bartonella species

Because conventional microbiological isolation techniques lack sensitivity, bartonellosis is usually diagnosed by PCR amplification of organism specific DNA sequences and/or through serological testing. Recently, the development of a more sensitive enrichment culture approach, using BAPGM (Bartonella alpha Proteobacteria growth medium) followed by real time PCR has greatly facilitated the molecular detection or isolation of Bartonella species from the blood of sick or healthy animals, including dogs, horses and human beings.5,6,12 Obviously, the relative sensitivity of the diagnostic methods used to detect Bartonella species infection greatly influences an investigator’s ability to establish disease causation or a clinicians ability to initiate appropriate treatment. Specifically, the use of this optimized microbiological approach has facilitated the recognition of blood-borne Bartonella spp. infections in dogs, horses, human beings and porpoises.17 Diagnostic testing (animals and humans) for Bartonella species (serology, PCR and BAPGM Enrichment Blood Culture/PCR) is available through Galaxy Diagnostics, Inc. (contact@galaxydx.com). In cats, serology, PCR or culture combined with serology is recommended and can be procured at Galaxy Diagnostics Inc. and Colorado State University (www.dlab.colostate.edu)

Pathologic Findings In dogs (and humans), pathologic findings associated with Bartonella spp. infection include endocarditis, myocarditis, granulomatous lymphadenitis, granulomatous hepatitis, osteomyelitis, bacillary angiomatosis and peliosis hepatitis.3,9,12,13 Multifocal areas of severe myocardial inflammation can be found in dogs with B. vinsonii (berkhoffii) endocarditis. Although not specific for bartonella infections, organisms can be detected in diseased tissues using silver stains, particularly in acute bartonella infections, such as acute regional lymphadenitis (cat scratch disease). During chronic infections, organisms are often too few in number to be detected in tissues by silver staining, unless a fulminate infection is localized to heart valves. The cardiac abnormalities noted in cats to date are similar to those described for dogs.9 It seems likely that the spleen plays an important immunomodulatory role in controlling persistent Bartonella spp. bacteremia in animals and people.13 The extent to which Bartonella spp. induce splenic pathology deserves additional research consideration. Therapy

To date, an optimal protocol has not been established for the treatment of bartonella infections in cats, dogs, or people.3,17 Regardless of the antibiotic(s) that is used for treatment, a long duration of antibiotic administration (at least 4-6 weeks) may be necessary to eliminate the infection. Due to the rapid development of resistance to macrolides (azithromycin), I no longer recommend these antibiotics as a sole or first-line antibiotic for treating Bartonella infections. Fluoroquinolones in combination with doxycycline are currently being used by the author to treat clinical cases of bartonellosis, while exploring antibiotic efficacy following natural or experimental infections. Doxycycline alone does not appear to eliminate B. vinsonii (berkhoffii), B. henselae or B. clarridgeae in cats, dogs or other animal species. Serum antibody titers often decrease rapidly (3-6 months) and are generally no longer detectable in dogs that recover following antimicrobial therapy. Therefore, post-treatment serology may be a useful adjunct to BAPGM/PCR to determine if therapeutic elimination of bartonella infections has been achieved. Whether there is clinical benefit to follow serologic or molecular assay results in cats has not been widely studied, but most treated cats do not become seronegative in the short term. However, bacteremia can resolve after treatment or resolve spontaneously in some cats, whereas other cats remain bacteremic despite four to six weeks of antibiotic (documented for several antibiotic regimens) administration, despite resolution of clinical abnormalities (such as lethargy, inappetance and fever). Prevention

Although somewhat circumstantial, there is increasing evidence that Bartonella species can be transmitted by fleas and ticks to cats, dogs and human beings.17 Based upon scientific evidence generated during the past several decades, vector-transmitted pathogens can induce clinical manifestations ranging from acute fatal illness (i.e. Rocky Mountain spotted fever, ehrlichiosis, babesiosis and bartonellosis) to chronic debilitating disease states (ehrlichiosis, babesiosis, borreliosis, and bartonellosis). Therefore, minimizing or eliminating flea and tick exposure is perhaps of greater veterinary and public health importance today, than during any previous time in history. When rigorous flea and

tick control measures are instituted, it is highly probable that transmission of Bartonella species to pets and their owners will be greatly reduced or eliminated.18

Public and Occupational Health Considerations There is increasing evidence to support an important role for Bartonella species as a cause of a spectrum of disease manifestations in human patients.4,17,19-25 Due to extensive contact with a variety of animal species, veterinary professionals appear to be at occupational risk for infection because of frequent exposure to Bartonella spp., therefore these individuals should exercise increased precautions to avoid arthropod bites, arthropod feces (i.e. fleas and lice), animal bites or scratches and direct contact with bodily fluids from sick animals.26 As Bartonella spp. have been isolated from cat, dog or human blood, cerebrospinal fluid, joint fluid, aqueous fluid, seroma fluid and from pleural, pericardial and abdominal effusions, a substantial number of diagnostic biological samples collected on a daily basis in veterinary practices could contain viable bacteria. The increasing number of defined Bartonella spp., in conjunction with the high level of bacteremia found in reservoir-adapted hosts, which represent the veterinary patient population, ensures that all veterinary professionals will experience frequent and repeated exposure to animals harboring these bacteria.27,28 Therefore, personal protective equipment, frequent hand washing and avoiding cuts and needle sticks have become more important as our knowledge of this genus has improved and various modes of transmission have been defined. Physicians should be educated as to the large number of Bartonella spp. in nature, the extensive spectrum of animal reservoir hosts, and the diversity of confirmed and potential arthropod vectors, current limitations associated with diagnosis and treatment efficacy, and the ecological and the medical complexities induced by these highly evolved intravascular, endotheliotropic bacteria. References: 1. Breitschwerdt EB, DL Kordick: Bartonella infection in animals: carriership, reservoir potential, pathogenicity, and zoonotic potential for human infection. Clin Microbiol Rev 2000;13:428–438. 2. Chomel BB, RW Kasten, JE Sykes, H-J Boulouis, EB Breitschwerdt: Clinical impact of persistent Bartonella bacteremia in humans and animals. Annals New York Academy of Sciences 2003;990:1-12. 3. Breitschwerdt EB, Maggi RG, Chomel BB, Lappin MR. Bartonellosis: An emerging infectious disease of zoonotic importance to animals and to human beings. J Vet Emerg Crit Care 2010;20:8-30. 4. Breitschwerdt EB, Sontakke S, Hopkins S. Neurological manifestations of bartonellosis in immunocompetent patients: A composite of reports from 2005-2012. J Neuroparasitology 2012=;:3: 1-15. 5. Mascarelli PE, Maggi RG, Hopkins S, Mozayeni BR, Trull CL, Bradley JM, Hegarty BC, Breitschwerdt EB. Bartonella henselae infection in a family experiencing neurological and neurocognitive abnormalities after woodlouse hunter spider bites. Parasites and Vectors 2013;6:98. 6. Maggi RG, Ericson M, Mascarelli PE, Bradley JM, Breitschwerdt EB. Bartonella henselae bacteremia in a mother and son exposed to ticks in the Netherlands. Parasites and Vectors 2013;6:101. 7. Maggi RG, Mascarelli PE, Havenga LN, NAidoo V, Breitschwerdt EB. Co-infection with Anaplasma platys, Bartonella henselae and Candidatus Mycoplasma hematoparvum in a veterinarian. Parasites and Vectors 2013;6:106. 8. Balakrishnan N, Jawanda JS, Miller MB, Breitschwerdt EB. Bartonella henselae infection in a man with hypergammaglobulinaemia, splenomegaly and polyclonal plasmacytosis. J Med Microbiol. 2013;62:338-41. 9. Chomel BB, RW Kasten, C Williams, AC Wey, JB Henn, R Maggi, S Carrasco, J Maxet, HJ Boulouis, R Maillard, EB Breitschwerdt. Bartonella Endocarditis: A pathology shared by animal reservoir hosts and patients. Ann NY Acad Sci 2009;1166:120-126. 10. Henn JB, Liu CH, Kasten RW, VanHorn BA, Beckett LA, Kass PH, Chomel BB. Seroprevalence of antibodies against Bartonella species and evaluation of risk factors and clinical signs associated with seropositivity in dogs. Am J Vet Res. 2005;66:688-94. 11. Breitschwerdt EB, Linder KL, Day MJ, Maggi RG, Chomel BB, Kempf VAJ. Koch’s Postulates and the Pathogenesis of Comparative Infectious Disease Causation Associated with Bartonella species. J Comp Pathol 1: 1-11, 2013 12. Perez C, Diniz PPVP, Maggi RG, Breitschwerdt EB. Molecular and serologic diagnosis of Bartonella infection in 61 dogs from the United States. J Vet Intern Med 2011;25:805-810. 13. Varanat M, Maggi RG, Linder KE, Breitschwerdt EB. Molecular prevalence of Bartonella, Babesia and hemotropic Mycoplasma sp. in dogs with splenic disease. JVIM 2011;25:1284–1291. 14. Beerlage C, Varanat M, Linder K, Maggi RG, Colley J, Kempf AJK, Breitschwerdt EB. Bartonella vinsonii subsp. berkhofffii and Bartonella henselae as potential causes of proliferative vascular disease in animals. Med Microbiol Immunol. Med Microbiol Immunol. 2012: 319-326. 15. Pérez Veraa C, Diniz PPVP, Pultorak EL, Maggi RG, Edward B. Breitschwerdt EB. An unmatched case controlled study of clinicopathologic abnormalities in dogs with Bartonella infection. Comp Immunol Microbiol Infect Dis 2013;36:481-487. 16. Whittemore JC, Hawley JR, Radecki SV, Steinberg JD, Lappin MR. Bartonella species antibodies and hyperglobulinemia in privately owned cats. J Vet Intern Med. 2012;26:639-44. 17. Breitschwerdt EB. Bartonellosis: One health perspectives for an emerging infectious disease. ILAR Journal (Institute for Laboratory Animal Research, National Academy of Sciences) 2014;55:46-58. 18. Lappin MR, Davis WL, Hawley JR, Brewer M, Morris A, Stanneck D. A flea and tick collar containing 10% imidacloprid and 4.5% flumethrin prevents flea transmission of Bartonella henselae in cats. Parasit Vectors. 2013;25:26. 19. Breitschwerdt EB, PE Mascarelli, LA Schweickert, RG Maggi, BC Hegarty, JM Bradley, CW Woods: Hallucinations, sensory neuropathy, and peripheral visual deficits in a young woman infected with Bartonella koehlerae. J Clin Microbiol 2011;49:3415-3417. 20. Cherry NA, Maggi RG, Rossmeisl JH, Hegarty BC, Breitschwerdt EB. Ecological Diversity of Bartonella Species Infection among Dogs and Their Owner in Virginia. Vector Borne Zoonotic Dis 2011;11:1425-32. 21. Maggi RG, Mascarelli PE, Pultorak EL, Hegarty BC, Bradley JM, Mozayeni BR, Breitschwerdt EB. Bartonella spp. bacteremia in high-risk immunocompetent patients. Diagn Microbiol Infect Dis. 2011;71:43-37. 22. Breitschwerdt EB, RG Maggi, B Sigmon, WL Nicholson: Isolation of Bartonella quintana from a woman and a cat following putative bite transmission. J Clin Microbiol 2007;45:270-272. 23. Breitschwerdt EB, Maggi RG, Farmer P, Mascarelli PE. Molecular evidence of perinatal transmission of Bartonella vinsonii subsp. berkhoffii and B. henselae to a child. J Clin Microbiol. 2010;48:2289-2293. 24. Oliveira AM, RG Maggi, CW Woods, EB Breitschwerdt EB: Putative needle stick transmission of Bartonella vinsonii subsp. berkhoffii to a veterinarian. J Vet Intern Med. 2010;24:1229-1232.

25. Rossi MA, Balakrishnan N, Linder KE, Messa JB, Breitschwerdt EB. Concurrent Bartonella henselae infection in a dog with panniculitis and owner with ulcerated nodular skin lesions. Vet Dermatol In press. 26. Balakrishnan N, Pritchard J, Ericson M, Grindem C, Phillips K, Jennings S, Matthews K, Tran H, Birkenheuer AJ, and Breitschwerdt EB. Prostatitis, steatitis and eosinophilic enteritis in a dog following presumptive flea transmission of Bartonella henselae. J Clin Microbiol 2014;52:3447-3452. 27. Lantos PM, Maggi RG, Ferguson B, VarkeyJ, Park LP, Breitschwerdt EB, Woods CW. Detection of Bartonella species in the blood of veterinarians and veterinary technicians: A newly recognized occupational hazard. Vector Borne Zoonotic Dis Dis 2015;14:563-570. 28. Ericson M, Balakrishnan N, Mozayeni BR, Woods CW, Dencklau J, Kelly S, Breitschwerdt EB. Culture, PCR, DNA sequencing, and second harmonic generation (SHG) visualization of Bartonella henselae from a surgically excised human femoral head. Clinical Rheumatology 2017.

UPDATE ON CANINE AND FELINE ANAPLASMOSIS AND EHRLICHIOSIS Edward B. Breitschwerdt DVM, Diplomate ACVIM

College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA

INTRODUCTION Tick-borne pathogens are of tremendous historical importance to both veterinary and

human medicine. Recent events emphasize an expanding role for newly discovered, as well as previously recognized tick-transmitted organisms, as a cause of animal and human suffering. One of the most important new developments related to ehrlichiosis is the realization that a given mammalian species can be infected simultaneously or sequentially by several Ehrlichia species.1 As an example, both dogs and people can be infected with Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia ewingii, Ehrlichia muris Panola Mountain Ehrlichia, Anaplasma platys and Anaplasma phagocytophilium. During the past decade observations related to ehrlichiosis in animals have contributed substantially to the rapid expansion of new knowledge related to human anaplasmosis and ehrlichiosis.2,3 Increasingly, veterinarians in practice are being called upon to provide comparative medical information about ehrlichiosis in animals and to discuss the zoonotic risks that are attributable to members of the genus Ehrlichia. Without question, the increased spectrum of human and companion animal recreational activities continue to bring each of us, as well as our pets, into contact with competent tick vectors. Therefore, so as to decrease disease transmission, drug manufacturers should continue to search for effective acaricides and products with strong repellent characteristics, so as to prevent tick attachment and the need to treat ehrlichiosis in pets. Veterinarians play a critically important Public Health role in the context of vector-borne infectious diseases throughout the world. COMPARATIVE MEDICAL IMPORTANCE OF ANAPLASMA AND EHRLICHIA SPECIES INFECTIONS.

Of comparative medical interest, cats, dogs, humans, as well as other domestic and wild animal species, can all be infected with the same Anaplasma or Ehrlichia sp. For example, E. chaffeensis has been shown to infect dogs, goats, deer, and human beings.3,5 Similarly, A. phagocytophilium can induce similar disease manifestations in cats, dogs, horses and human beings and has alos be detected in blood samples from a wide range of wild animals. With the recent application of new molecular diagnostic techniques, the study of vector-borne disease problems has been enhanced. This technology continues to result in substantial clarification of the role of established agents in the pathogenesis of previously undocumented disease sequelae. In many respects, the immunopathogenic consequences of tick-borne infections, such as anaplasmosis and ehrlichiosis, are nearly identical among infected animal species and human patients. Often, the experimental characterization of the immunopathological response of a specific Ehrlichia sp. in animals has provided important insights as to the potential pathogenic consequences induced when the same organism infects human patients. Conversely, observations in human patients have contributed to the recognition of an increased spectrum of disease manifestations in animals, such as acute renal failure or acute respiratory distress syndrome (ARDS) in dogs infected with Ehrlichia sp.

Feline Anaplasmosis and Ehrlichiosis

In general, our knowledge of tick-borne diseases in cats is substantially less than our knowledge of the comparable disease in dogs or human patients. Recent molecular evidence indicates that cats can be infected with A. phagocytophilium and an E. canis-like organism. The infrequent diagnosis of ehrlichiosis in cats may be related to a number of factors including: a general under-recognition of tick-borne diseases in cats, decreased pathogenicity of tick-borne pathogens in cats as compared to other animals, or the more rapid removal of ticks from cats resulting in decreased opportunity for disease transmission. Most tick-transmitted pathogens require a 24 to 48 hour period of attachment to the host before there can be successful transmission of infectious organisms. Fastidious grooming may result in the early removal of most ticks from cats and thereby the prevention of disease transmission.

Although various Ehrlichia, Anaplasma and Neorickettsia species have been reported to cause disease in cows, sheep, dogs, horses and human beings, the role of any specific species as a pathogen in cats remains less clearly defined. The first evidence for naturally-occurring feline ehrlichiosis was provided by Charpetier and Groulade in France. Feline ehrlichiosis was subsequently reported in 1989, by Buoro and colleagues, when they described intracytoplasmic inclusions in monocytes and lymphocytes derived from 3 cats in Kenya. By both light and electron microscopy, the inclusions were morphologically similar to Ehrlichia sp. morulae, as observed on blood smears obtained from other animals. Subsequently, morulae were described in stained blood smears obtained from cats in the United States, France, Brazil and Sweden. To date, no Anaplasma or Ehrlichia species has been cultured from the blood of a cat. Bjoersdorff and colleagues amplified and sequenced 16S rDNA from an EDTA blood sample obtained from a 14-month-old shorthaired cat from Sweden that was 100% similar to canine and equine A. phagocytophilium strains from the same region.

In conjunction with Dr. Mike Lappin at Colorado State University, we have amplified and sequenced A. phagocytophilum DNA from blood samples or cats from Ixodes scapularis endemic regions. We have also amplified A. phagoctyophilum DNA from a small number of cats from the southeastern United States that had hematological abnormalities (non-regenerative anemia, thrombocytopenia, or pancytopenia).

Previously, our research group described E. canis-like infection in young cats from the southeastern United States, eastern Canada and France. Based upon PCR amplification and DNA sequencing, the Ehrlichia DNA amplified from the blood of these cats was 100% similar to the comparable E. canis DNA sequences obtained form canine E. canis isolates. As no isolates were made from these cats, a more complete genetic characterization was not possible. We are currently describing these feline infections as E. canis-like, particularly as antibodies could not be detected in these cats by IFA testing using E. canis antigens. Interestingly, serum from all 3 cats in our study contained anti-nuclear antibodies. The predominant disease manifestations were polyarthritis, accompanied by fever in 1 cat, bone marrow hypoplasia or dysplasia, accompanied by pancytopenia in a second cat and anemia and thrombocytopenia in the third cat. (Figure 2) In dogs, neutrophilic polyarthritis has been most frequently associated with E. ewingii infection. In this cat neutrophilic polyarthritis was confirmed by cytologic analysis of joint fluid at 1 and 3 years of age, suggesting the possibility of chronic E. canis-like infection. Previous serologic studies, have reported an association between thrombocytopenia, hyperglobulinemia and polyarthritis in cats that had E. canis antibodies. Other nonspecific clinical abnormalities, including lethargy, anorexia, conjunctivitis, swelling in the ventral neck region and mild interstitial lung disease were reported in cats and can be observed in association with canine ehrlichiosis.

Canine ehrlichiosis is an infectious rickettsial disease of dogs, caused by E. canis, E.

chaffeensis, E. ewingii, E. muris Panola Mountain Ehrlichia and potentially E. ruminantium. Although the clinicopathologic course of disease will vary depending upon the infecting Ehrlichia species, illness is typically characterized by an acute reduction in cellular blood elements, most often thrombocytopenia. Canine ehrlichiosis, caused by E. canis, has been reported from tropical and subtropical regions throughout the world. The distribution of E. canis infection is related to the geographic distribution of the vector tick, Rhipicephalus sanguineous, the brown dog tick, which spends all 3 life stages on dogs. Canine ehrlichiosis, caused by E. chaffeensis and E. ewingii, have been diagnosed in the United States and Africa. Amblyomma americanum, the Lone Star tick, is the most important vector for E. chaffeensis and E. ewingii in North America. Co-infection with multiple Ehrlichia species or Ehrlichia and Anaplasma spp. is not uncommon. Based upon experimental infection studies, canine ehrlichiosis has been divided into 3 phases: an acute, subclinical and chronic disease phase. Although these 3 phases of disease can be utilized to infer some clinical utility, the onset and duration of infection is rarely known in the clinical setting. Clinical signs during the acute phase of disease are highly variable and can include: depression, anorexia, fever, severe loss of stamina, weight loss, ocular and nasal discharges, dyspnea, lymphadenopathy, and edema of the limbs or scrotum.. Thrombocytopenia and leukopenia

generally occur 10 to 20 days following infection. Despite moderate to severe thrombocytopenia, hemorrhages are rarely observed. A variety of central nervous system signs, including hyperesthesia, muscle twitching, and cranial nerve deficits, may occur due to inflammation and bleeding into the meninges. Clinical findings in the acute phase of ehrlichiosis can be identical to canine Rocky Mountain spotted fever or canine distemper. Serologic diagnosis utilizing the indirect fluorescent antibody technique (IFA) is currently recommended for confirming a diagnosis of ehrlichiosis. 25 The IFA test for E. canis is sensitive and reasonably specific; however, based upon Western immunoblot (WI) analysis, low IFA titers are not diagnostic and may represent exposure to other infectious organisms. Current modalities that detect E. canis antibodies in serum samples obtained from dogs for diagnostic purposes, such as the microimmunofluorescent assay (IFA), do not facilitate differentiation of the infecting Ehrlichia species. There is substantial serologic cross reactivity between E. canis and E. chaffeensis, whereas E. ewingii infected dogs generally do not recognize E. canis antigens or do so at very low titers. Dogs generally become seronegative within 3 to 9 months after effective treatment, although some dogs maintain persistent and stable titers for years. Polymerase chain reaction (PCR) amplification can facilitate a molecular confirmation of the diagnosis of canine ehrlichiosis, determine of the infecting Ehrlichia species or help to confirm the therapeutic elimination of infection. EDTA blood is required for PCR and should optimally be collected for diagnostic confirmation prior to antibiotics or to confirm therapeutic elimination of infection after cessation of antibiotics. Tetracycline (22 mg/kg given every 8 hours) or doxycycline (5 mg/kg every 12 hours), administered daily for 4 weeks, represent the treatment of choice for canine and feline anaplasmosis and ehrlichiosis. Clinical improvement may be observed in E. canis infected dogs with penicillin, sulfonamides, enrofloxacin or imidocarb dipropionate but the therapeutic response is incomplete and therefore these antibiotics cannot be recommended. Dramatic clinical improvement generally occurs within 24 to 48 hours after initiation of a tetracycline derivative in dogs with acute phase or mild chronic phase disease. Hemorrhage, immunosuppression and concurrent infections with Babesia or Bartonella species may contribute to the death of chronically affected dogs, despite the initiation of tetracycline therapy. The duration of treatment of chronically affected dogs with severe pancytopenia or aplastic anemia is controversial. Despite clinical improvement and presumable clearance of the infection, bone marrow regeneration may require up to 120 days following treatment. Supportive therapy, including fluids, blood transfusion, vitamins, and anabolic steroids are required in some patients. Long-term tetracycline prophylaxis (6.6 mg/kg once daily), repositol oxytetracycline (200 mg IM twice weekly) or doxycycline have been utilized in military working dogs or dogs maintained in tick infested kennels to prevent ehrlichiosis during deployment to tick endemic regions. Following therapeutic elimination of Ehrlichia spp., dogs do not develop protective immunity and can be re-infected when re-introduced to a vector-competent tick. Experimentally, dogs have been re-infected with E. canis by both homologous and heterologous challenge. Although not well characterized, the long term prognosis following treatment for ehrlichiosis does not appear to be predictable. The reasons for variability in post-treatment outcomes in dogs with ehrlichiosis remains to be established through long term follow-up studies. ZOONOTIC IMPLICATIONS OF EHRLICHIOSIS Based upon isolation from patients, E. canis, E. chaffeens, E. ewingii, E. muris and Panola Mountain Ehlrlichia can all cause human ehrlichiosis. However, the zoonotic role of dogs as a reservoir for human infection has not been clearly established for any Ehrlichia species. In South America, E. canis causes human monocytic ehrlichiosis and dogs are the probable reservoir host. Tick control is critically important, but does not assure prevention of the disease or elimination of re-infection. Although not well characterized, the long term prognosis following treatment for ehrlichiosis does not appear to be predictable The reasons for variability in post-treatment outcomes, particularly in dogs with ehrlichiosis, remains to be established through long term follow-up studies.

Selected References:

1. Kordick SK, Breitschwerdt EB, Hegarty BC, et al. Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina. J Clin Microbiol 1999; 37:2631-2638.

2. Breitschwerdt EB, Hegarty BC, Hancock SI. Doxycycline hyclate treatment of experimental canine ehrlichiosis followed by challenge inoculation with two Ehrlichia canis strains. Antimicrob Agents Chemother 1998; 42:362-368.

3. Frank JR, Breitschwerdt EB. A retrospective study of ehrlichiosis in 62 dogs from North Carolina and Virginia. J. Vet Intern Med 1999;13:194-201.

4. Breitschwerdt EB, Hegarty BC, BA, Qurollo BA, Saito TB, Maggi RG, Blanton LS, Bouyer BH. Intravascular persistence of Anaplasma platys, Ehrlichia chaffeensis, and Ehrlichia ewingii DNA in the blood of a dog and two family members. Parasit and Vect. 2014;7:298.

5. Maggi RG, Mascarelli PE, Havenga LN, NAidoo V, Breitschwerdt EB. Co-infection with Anaplasma platys, Bartonella henselae and Candidatus Mycoplasma hematoparvum in a veterinarian. Parasites and Vectors 6:106, 2013.

6. Hegarty BC, Qurollo BA, Thomas B, Park K, Chandrashekar R, Beall MJ, Thatcher B, Breitschwerdt EB. Serological and molecular analysis of feline vector-borne anaplasmosis and ehrlichiosis using species-specific peptides and PCR. Parasit Vectors. 2015;8:320.

7. Qurollo BA, Davenport AC, Sherbert BM, Grindem CB, Birkenheuer AJ, Breitschwerdt EB. Infection with Panola Mountain Ehrlichia sp. in a dog with atypical lymphocytes and clonal T-cell expansion. J Vet Intern Med. 2013 Sep-Oct;27(5):1251-5.

8. Qurollo BA, Chandrashekar R, Hegarty BC, Beall MJ, Stillman BA, Liu J, Thatcher B, Pultorak E, Cerrito B, Walsh M, Breitschwerdt EB. A serological survey of tick-borne pathogens in dogs in North America and the Caribbean as assessed by Anaplasma phagocytophilum, A. platys, Ehrlichia canis, E. chaffeensis, E. ewingii, and Borrelia burgdorferi species-specific peptides. Infect Ecol Epidemiol. 2014 Oct 20;4.

BORRELIOSIS: Of Cats, Dogs and Humans

Edward B. Breitschwerdt, DVM, Dipl. ACVIM

Comparative Medicine Institute

Department of Clinical Sciences, College of Veterinary Medicine

North Carolina State University, Raleigh, NC 27606

Veterinarians play a central role in the diagnosis, treatment and prevention of tick-

transmitted infectious diseases of companion animals. Veterinarians also play an

increasingly important role in advising the public as to the zoonotic potential of

organisms that are transmitted from ticks to pets or to their owners.

Lyme disease or borreliosis is a tickborne spirochetal disease of dogs and people that is

characterized primarily by oligoarticular arthritis. Canine borreliosis is caused by the same

spirochete (Borrelia burgdorferi) that is responsible for Lyme disease in human beings.

The human disease was first recognized in Lyme, Connecticut in 1975, and is characterized

by an expanding annular skin lesion called erythema chronicum migrans (ECM). To date,

an ECM lesion has not been described in a dog. Fever, weakness, headache, myalgia, and

arthralgia frequently accompany the acute phase of the human disease, and similar clinical

signs can be found in infected dogs. Days to months after the initial untreated infection,

recurrent arthritis, meningoencephalitis, peripheral neuropathies, myocarditis, or

atrioventricular conduction defects develop as immunologically or spirochete-mediated

aspects of chronic disease. In addition to arthritis, myocarditis and glomerulonephritis have

been related to borreliosis in the dogs.

Canine borreliosis was first reported in a dog from Long Island, New York in 1984.

Subsequently, a serosurvey, involving tick-infested regions of southern Connecticut,

identified a similar geographic clustering of dogs with antibodies to B. burgdorferi as the

distribution of human Lyme disease. In the United States, most canine and human cases

have been reported in the northeast, midwest (Minnesota, Wisconsin), the northern west

coast, and, more recently, in mid-Atlantic and southeastern states. The disease in human

beings has been reported from at least 36 states, Canada and Europe. Currently, there is a

major controversy as to whether Lyme disease occurs in Australia. Lyme disease is now

considered the most prevalent tickborne disease in the world. In North America, the

distribution of the disease, in both dogs and man, is related to the distribution of the vector

tick Ixodes scapularis or I. pacificus. It is possible that Amblyomma americanum

contributes to transmission of a borrelial species (not B. burgdorferi) in the southeastern

United States. Transplacental transmission has been documented in pregnant women, but

based upon experimental studies in utero is unlikely to occur in dogs.

The organism, B. burgdorferi, is very difficult to culture, even with the use of specialized

media. In humans, the spirochete has been rarely cultured from blood, joint fluid or

cerebrospinal fluid, but B. burgdorferi can be more readily cultured from skin, particularly

at the site of tick attachment. Obviously, culturing skin biopsies has only limited utility for

humans with ECM lesions and no utility for companion animal diagnosis, as the site of tick

attachment(s) may or may not be known and ECM lesions have not been reported. PCR

amplifications of B. burgdorferi DNA can be used to support a diagnosis of Lyme arthritis

or neuroborreliosis. The duration of spirochetemia in dogs following natural infection is

unknown, however, spirochetes can be isolated from tick attachment sites and less

frequently joint fluids for periods up to 1 year following experimental infection. Borrelia

burgdorferi appears to share only minor antigenic relatedness to leptospiral organisms, but

there is serologic cross-reactivity with relapsing fever spirochetes, when using indirect

fluorescent antibody testing as a diagnostic modality, which is no longer a recommended

approach. Borrelia burgdorferi can infect a variety of vertebrates (birds, dogs, deer, mice,

and others); however, despite the apparent broad host range, disease has been conclusively

documented only in dogs and human beings. Although cats become infected and develop

B. burgdorferi-specific antibodies, it remains unclear if and how often cats develop disease

manifestations. In most clinical reports, disease manifestations in cats, cows, dogs and

horses have been temporally associated with serologic evidence of B. burgdorferi infection.

Serology can only implicate exposure and because of the high seroprevalence in healthy

animals in the regions from which these reports have originated our ability to determine

the clinical relevance (disease causation) of these reports is compromised.

The most compelling evidence related to the pathogenicity of B. burgdorferi in dogs

originated from Dr. Max Appel’s laboratory at Cornell University. In experimental tick

attachment studies using dogs, adult Ixodes scapularis (dammini) ticks were more much

likely to induce disease signs than were larval ticks (the most frequent source of B.

burgdorferi transmission to humans). Consistent with previous studies, including those in

our laboratory, injection of cultured organisms, by a variety of routes, resulted in antibody

production, but no clinical signs in the infected dogs. Experimentally, as has been

documented in rodent studies, infection of nonimmunocompromised dogs with

B. burgdorferi appears to be age dependent (i.e., puppies are more susceptible and more

likely to develop lameness than adult dogs) and there are clearly breed differences in

disease expression. For example, “Lyme” nephritis is most often reported in Golden

retrievers or Labrador retrievers. Based upon experimental infection studies involving

beagles, there are even within breed differences in the propensity to develop disease

manifestations.

Presumably, immunocompetence at the time of tick attachment is an important determinant

for the development of disease signs. In Dr. Appel’s tick attachment studies, 11/14 six-

week-old puppies developed lameness, 3/16 twelve-week-old puppies became lame, and

0/5 six-month-old dogs developed lameness. Additionally, 2 bitches infected during

pregnancy failed to develop clinical signs and their puppies did not become infected.

Current clinical and experimental studies indicate that only a small percentage of dogs

exposed to B. burgdorferi develop clinical signs of arthritis or other Lyme disease

manifestations. Extrapolation from the human literature suggests that the development of

disease in dogs may correlate with a specific histocompatibility type, concurrent illness, or

congenital or acquired immunodeficiency states. Variation in breed susceptibility deserves

additional study, particularly in the context of Lyme nephritis.

Arthritis is associated with the acute and chronic phases of disease in humans. It is not clear

if arthritis in dogs is related to an acute or chronic manifestation of the disease, but

persistent infection for months and perhaps years has been supported by serologic culture

and PCR evidence. Most dogs do not seroconvert to B. burgdorferi until six weeks

following tick transmission; therefore serological testing following a recent tick exposure

is only useful to document the lack of antibodies. Convalescent serological testing, after

six weeks, can be used to document seroconversion. Retrospective serologic evidence

suggests that arthritis can occur in both acute and chronic phases of canine borreliosis.

Localization of circulating immune complexes in tissues, such as the synovium, is thought

to result in a lymphocytic-plasmacytic synovitis that is responsible for the chronic arthritic

manifestations of the disease.

Anorexia, depression, fever, stiffness, joint pain, and swelling are reported features of

canine borreliosis; however, it is important to realize that Anaplasma phagocytophilum,

which can be co-transmitted with B. burgdorferi by Ixodes species ticks throughout the

Northern Hemisphere induces identical clinical signs. Disease occurs in dogs from regions

endemic for human Lyme disease, with the onset of clinical signs most often during the

spring and summer months. In dog experimental infection tick attachment studies clinical

signs include: lameness that resolved spontaneously and rarely exceeded 4 days in duration,

fever, and infrequently, mild lymphadenopathy with observation periods of up to 17

months in duration. Again, anaplasmosis has been concurrently documented in many of

these tick attachment studies. Histologically, cellular infiltrates have been limited to the

joints (fibrinopurulent or lymphocytic/ plasmacytic arthritis), skin, and lymph nodes and

no lesions were found in the heart, brain or other tissues. During the initial 6–8 weeks

following experimental tick attachment studies, most dogs develop a second episode of

lameness, and some dogs a third episode, after which no dog (all studies used beagles)

developed additional signs during the remaining portion of the observation periods. A

lesion similar to erythema chronicum migrans has not been associated with borreliosis in

dogs that are naturally or experimentally infected. Additional findings from Dr. Appel’s

studies suggest that environmental transmission of B. burgdorferi through urine or direct

contact is highly unlikely. Based on xenodiagnostic results (placing ticks on B. burgdorferi

infected dogs), dogs appear to pose a very minor or no reservoir potential for facilitating

the spirochete transmission in nature. Thus, an infected dog poses minimal to no risk to

family members.

Following tick attachment and B. burgdorferi transmission, the incubation period prior to

the development of lameness ranges from 2–5 months. Seroconversion occurs by 6 weeks

following tick attachment, and can be documented frequently in dogs that do not exhibit

signs of disease. Experimentally, all dogs from which B. burgdorferi was cultured and/or

disease signs developed seroconverted. This observation suggests that a diagnosis of Lyme

disease in a seronegative dog would seem unlikely. Seronegative “Lyme disease” remains

controversial in human medicine.

Experimentally, despite a lack of clinical signs, B. burgdorferi can be cultured from the

site of tick attachment at least 9 months following infection, clearly supporting the presence

of viable spirochetes long after initial tick exposure. The extent to which chronic

persistence of the spirochete eventually contributes to myocarditis, encephalitis, immune-

mediated, or other disease manifestations in dogs requires additional study. Long-term

persistence of B. burgdorferi in clinically healthy dogs suggests the possibility of

premunition, and when considered in conjunction with exposure to less virulent strains of

the organism, might explain the high seroprevalence (as high as 90% in dogs from

Connecticut when Lyme disease was first being studied) to B. burgdorferi in healthy dogs

in endemic areas. We have also documented the persistence of antibodies (up to 4 years in

our experience) in a healthy dog that moved from an endemic (Connecticut) to a

nonendemic area (North Carolina). It should be emphasized that canine seroprevalence

results from our laboratory indicate that exposure to B. burgdorferi in North Carolina is

during the period 1983–2004 was very infrequent or non-existent. Thus, for over two

decades serologic data did not support the transmission of B. burgdorferi to dogs residing

in North Carolina. Over the past decade there has been a progressive southern movement

of B. burgdorferi transmission through Virginia into North Carolina. We now have B.

burgdorferi transmission predominantly in the coastal and central counties of North

Carolina. Borrelia burgdorferi transmission may be occurring in other focal sites in the

southeastern United States, but most data supports movement of infected dogs from

endemic to non-endemic locations.

With rare exceptions, hematologic or biochemical abnormalities (exception Lyme

nephritis) have not been reported in association with B. burgdorferi infection in dogs or

humans. If hematological abnormalities are found in a B. burgdorferi seroreactive dog,

concurrent infection with A. phagocytophilum or other vector borne pathogens should be

pursued diagnostically. Alternatively, the dog may have been exposed to B. burgdorferi

months to years earlier and is suffering from another infectious or non-infectious disease.

Joint radiographs, with the exception of mild distention of the joint capsule, are normal.

Clinically, joint involvement can be monarticular or oligoarticular. Synovial fluid analysis

can reveal high cell counts (46,000 neutrophils/mm at the onset of lameness) consisting

predominantly of neutrophils during the early stages of infection and mononuclear cell

infiltrates in association with chronic infection.

Diagnosis of Borrelia burgdorferi: The B. burgdorferi C-6 peptide in the SNAP 4DX test

(IDEXX Laboratories) is a highly specific diagnostic peptide that is used to detect B.

burgdorferi antibodies in dog sera. Infection with closely related bacteria or prior

vaccination with a “Lyme Disease” vaccine will not result in antibodies to the B.

burgdorferi C-6 peptide. Specificity of the test is exceptional and approaches 100%.

However, when used as an annual screening test, occasional false positive test results

should be anticipated. Questionable B. burgdorferi positive test results should be further

examined by Western immunoblot at Cornell University. IFA testing using whole cell B.

burgdorferi as antigen is not recommended because the specificity is poor compared to the

C-6 peptide assay and Lyme vaccination will result in IFA seroreactivity. WI analysis can

be used to differentiate vaccination from natural exposure. In endemic regions, numerous

clinically healthy dogs (up to 90% in highly endemic regions) have high IgM and IgG IFA

antibody titers to B. burgdorferi. Obviously, in the absence of documented seroconversion,

high seropositivity in endemic regions invalidates a serodiagnosis. Culture of the organism

and dark field microscopy of joint fluid have generally been an ineffective means of

substantiating a diagnosis. PCR is available for testing CSF or joint fluids, but is not

recommended for blood testing, as B. burgdorferi spirochetemia occurs only after the acute

infection.

Treatment: Although controversial, based upon current data, I do not recommend

routine treatment of C-6 antibody positive dogs, unless there is concurrent clinical

disease. (See the ACVIM Consensus Statement on Lyme Disease in Dogs) To date,

Lyme disease in dogs is characterized by lameness, which can be intermittent and self-

limiting without antibiotic administration. Less well documented abnormalities include

encephalitis, myocarditis and acute renal failure (particularly in Labrador and Golden

Retrievers). Although antibiotic treatment can enhance the resolution of lameness, no

antibiotic has been proven to be curative. If we do not cure the infection, treatment of a

healthy dog is more difficult to justify. Clearly, with the advent of new and more

efficacious vaccines, prevention of B. burgdorferi infection in Lyme endemic regions is

recommended.

Doxycycline is the treatment of choice (5 mg/kg given every 12 hours for 14 days), as this

antibiotic is effective for dogs co-infected with B. burgdorferi and A. phagoctyophilum.

Penicillin derivatives are also effective. Treatment of chronic arthritis, cardiac, or

neurologic signs in human patients has resulted in complete or partial remission of signs:

however, some individuals fail to improve following extended courses of antibiotics.

Unfortunately, the canine response to treatment has not been detailed in most clinical

reports. In contrast to humans with documented Lyme disease, antibiotic treatment does

not appear to induce a decrease in IFA antibody titers in dogs, as might be expected.

Following experimental infection, doxycycline and amoxicillin suppressed, but failed to

clear the infection. Although the quantitative C6 peptide has been recommended as a means

to confirm therapeutic elimination, data to confirm this recommendation is lacking.

Due to the extremely small size of ixodid ticks, in comparison to other ticks that frequently

infest dogs, detection is difficult. In addition, the smaller nymphal tick is thought to be

most important in human disease transmission and most people with Lyme disease do not

recall a tick bite. The only effective means of prevention is the routine use of safe and

effective acaracide products and the avoidance areas of high endemicity for the spirochete.

Lyme vaccination is recommended for dogs in endemic regions and for pets that will

vacation in endemic areas. In locally endemic areas, up to 60% of the ticks have been found

to be infected with B. burgdorferi, thus the possibility of transmission is high.

Selected References:

1. Duncan AW, MT Correa, JF Levine, Breitschwerdt EB: The dog as a sentinel for human

infection: Prevalence of Borrelia burgdorferi C6 antibodies in dogs from southeastern and

mid-Atlantic states. Vector Borne Zoonotic Dis 4:221-229; 2004.

2. O’Connor TP, Hanscom J L Hegarty B C, Groat RG, Breitschwerdt E B Comparison of

an indirect immunofluorescence assay, western blot analysis and a commercially available

ELISA for detection of Ehrlichia canis antibodies in canine sera. Am. J. Vet. Res. 2006;

67:206-210.

3. Solano-Gallego, L, Llull J, Osso M, Hegarty B, Breitschwerdt E. A serological study

of exposure to arthropod-borne pathogens in dogs from northeastern Spain. Vet Res.

2006;37:231-44.

4. Scott JD, Foley JE, Anderson JF, Clark KL, Durden LA. Detection of Lyme Disease

Bacterium, Borrelia burgdorferi sensu lato, in Blacklegged Ticks Collected in the Grand

River Valley, Ontario, Canada. Int J Med Sci. 2017;14(2):150-158.

5. Bjurman NK, Bradet G, Lloyd VK. Lyme disease risk in dogs in New Brunswick.

Can Vet J. 2016;57(9):981-4.

6.. Irwin PJ, Robertson ID, Westman ME, Perkins M, Straubinger RK. Searching for

Lyme borreliosis in Australia: results of a canine sentinel study. Parasit Vectors.

2017;10:114.

7. Oliver LD Jr, Earnhart CG, Virginia-Rhodes D, Theisen M, Marconi RT. Antibody

profiling of canine IgG responses to the OspC protein of the Lyme disease spirochetes

supports a multivalent approach in vaccine and diagnostic assay development. Vet J.

2016;218:27-33.

8. Feng J, Shi W, Zhang S, Zhang Y. Persister mechanisms in Borrelia burgdorferi:

implications for improved intervention. Emerg Microbes Infect. 2015;4(8):e51.