Gumshoe Sleuthing in the World of Infectious Disease and Neonatology: Discoveries That Changed Equine and Human Health

1. Introduction

I wish to thank the AAEP for the honor of delivering the Milne lecture at the 60th annual AAEP convention. The purpose of the Milne program as described by the AAEP is: “The lecture is intended to honor the accomplishments of the individual and bring a meaningful learning experience to the AAEP membership. The lecture should be a perspective on the state-of-the-art in your particular area of expertise.”

My career started as a private practitioner in Mendocino County in 1975 where I developed a rural veterinary practice for horses and built the first large animal hospital in the county. I have always identified with the equine practitioner in the field and private practice clinics. I returned to UC Davis in 1983 as an assistant professor. It was at UC Davis that I was given the freedom and opportunity to pursue discovery in the manner that I began in private practice. My investigation of disorders was a bit different, and more like detective work, than traditional epidemiology. Therefore, I am taking this once in a lifetime opportunity of the Milne lecture to share the investigative steps, the leads, the clues, the “thinking out of the box,” which led to discoveries in infectious disease and neonatology, which I have been requested to focus on in this presentation. Each section of the science of a topic will be preceded with the “Gumshoe Sleuthing” component for the readers, my fellow equine practitioners, who I hope can use this information for the pleasure of being who we are and for making a difference in the life of a horse or a foal.

“The joy is in creating, not maintaining.”
—Vince Lombardi, NFL Hall of Fame Coach

2. Equine Granulocytic Anaplasmosis

(Anaplasma phagocytophilum, Ehrlichia equi, The California Ehrlichia Agent)

Gumshoe Sleuthing

Gumshoe definition: Slang (1) An investigator, especially a detective. To move about stealthily. (2) To use the power of keen observation to obtain leads and develop ideas to solve mysteries. (3) A person whose job is to find information about someone or something.

When I graduated from veterinary school in 1975, there were 6 cases of the disease termed Ehrlichia equi, now known as Anaplasma phagocytophilum or Equine Granulocytic Anaplasmosis, worldwide. There were no known human cases, and all the diagnosed equine cases were in the sierra foothills of California. The first case seen at UC Davis was diagnosed by Racheal Smith, a hematology technician, who saw the unique and characteristic inclusion bodies in the white blood cells of a horse presented for persistent fever, ataxia, reluctance to
move, limb edema, and anorexia. The blood from the second horse, seen a year later, was transfused into a research horse that subsequently came down with a fever within 72 hours. The blood from that horse was frozen and used as a source for an extensive PhD project by David Gribble on the pathogenesis and pathology of the disease in horses. In the fall of 1975, 3 months after my graduation from veterinary school, I examined a 3-year-old horse with a fever, lethargy, ataxia, icterus, and mild petechiation in the parking lot of the small veterinary clinic where I was employed. I performed a complete blood count (CBC) in our laboratory and noted several characteristic inclusion bodies within neutrophils that were identical to the agent Gribble had identified. The horse had many ticks visible throughout the hair coat. The horse was treated with tetracycline; the fever defervesced within 24 hours and with 7 days of treatment the horse recovered. Two weeks later, I saw another case and had the inclusion bodies confirmed at UC Davis by sending the hematology slides to the lab. I traveled to various veterinary clinics in Mendocino and Humboldt County with the slides in my pocket showing other veterinarians, who expressed modest but polite interest. In 1981, thanks to accumulating many other horses diagnosed with A. phagocytophilum, I presented 41 cases at the AAEP meeting. I was hired by UC Davis in 1983 as an assistant professor coming directly from private practice to the University. In 1988, I began a research project with Dr. J. Stephen Dumler after the first human cases of granulocytic anaplasmosis were diagnosed by the same method of examining a blood film in a sick person who had tick exposure after a hike. By 1990, hundreds of human cases of what was then called Human Granulocytic Ehrlichia (HGE) were being diagnosed, with a significant number of fatalities from lack of prompt administration of tetracycline. The Center for Disease Control named HGE the second most common tick transmitted disease in the United States.

Copyright use authorized by Journal of Equine Veterinary Science: Pusterla N, Madigan JE. Equine granulocytic anaplasmosis. J Equine Vet Sci 2013;33:493–496.

Etiology

Anaplasma phagocytophilum (formerly Ehrlichia equi) is the etiologic agent of equine granulocytic anaplasmosis (EGA; formerly equine granulocytic ehrlichiosis). A. phagocytophilum has recently been classified, based on genetic analysis, in the genus Anaplasma with Anaplasma marginale, which causes infectious anemia in cattle by infecting erythrocytes, and Anaplasma platys, which causes canine cyclic thrombocytopenia by infecting platelets.¹

Because 16 S ribosomal ribonucleic acid (rRNA) gene sequences differ only up to 3 bases (99.1% homology) among former E. equi, Ehrlichia phagocytophila (cause of tick-borne fever in Europe), and the recently discovered HGE agent, these organisms are now all considered strains of A. phagocytophilum. E. equi, E. phagocytophila, and the HGE agent are also closely related on the basis of morphology, host cell tropism, and antigen analysis by indirect fluorescent antibody tests.² DNA sequences of the 16 S rRNA gene from the peripheral blood of naturally infected horses in Connecticut and California are identical with those of the HGE agent.³ Moreover, infected human blood from HGE patients injected into horses causes typical EGA, which can be transmitted to other horses. It induces protection in horses to subsequent challenge with A. phagocytophilum.^4,5

Anaplasma species are small (0.2–1.0 µm in diameter) obligate intracellular bacteria with a gram-negative cell wall⁶, but lack lipopolysaccharide biosynthetic machinery.⁷ The bacteria reside in an early endosome, where they obtain nutrients for binary fission and grow into a cluster called a morula. Genomic studies demonstrated a type IV secretion apparatus, which could facilitate transfer of molecules between the bacterium and the host.^8,9A. phagocytophilum is found within the cytoplasm of infected eukaryotic host cells, primarily neutrophilic and eosinophilic granulocytes. These inclusion bodies consist of one or more coccoid or coccobacillary organisms approximately 0.2 mm in diameter, as well as large granular aggregates called morulae, which are approximately 5 mm in diameter. Organisms are visible under high, dry, or oil-immersion objectives with light microscopy. They stain deep blue to pale bluish-gray with Giemsa or Wright–Leishman stain. Electron microscopy reveals loosely packed, ovoid to round A. phagocytophilum organisms in several membrane-lined vacuoles of equine granulocytes. The size of vacuoles ranges from 1.5 to 5 mm in diameter.

Epidemiology

Equine granulocytic anaplasmosis occurs during late fall, winter, and spring. The horse represents an aberrant host, and it seems unlikely that infected horses could serve as effective reservoirs of A. phagocytophilum because the presence of the organism in an affected animal is generally limited to the acute phase of the disease. Horses of any age are susceptible, but the clinical manifestations are less severe in horses younger than 4 years old.¹⁰ Horses from endemic areas have a higher seroprevalence of antibody to A. phagocytophilum than horses from nonendemic areas, suggesting the occurrence of subclinical infection in some animals.¹¹ [...]