Katherine A. Sayler

Diagnostic Hematology of Reptiles

The hematologic evaluation of reptiles is an indispensable diagnostic tool in exotic veterinary practice. The diversity of reptile species, their characteristic physiologic features, and effects of intrinsic and extrinsic factors present unique challenges for accurate interpretation of the hemogram. Combining the clinical presentation with hematologic findings provides valuable information in the diagnosis and monitoring of disease and helps guide the clinician toward therapy and further diagnostic testing. This article outlines the normal and pathologic morphology of blood cells of reptile species. The specific comparative aspects of reptiles are emphasized, and structural and functional abnormalities in the reptilian hemogram are described.

Seroprevalence of Ehrlichia canis, Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in North America

BackgroundThis study evaluated the exposure of dogs to three different Ehrlichia spp. in the south and central regions of the United States where vector-borne disease prevalence has been previously difficult to ascertain, particularly beyond the metropolitan areas.MethodsDog blood samples (n = 8,662) were submitted from 14 veterinary colleges, 6 private veterinary practices and 4 diagnostic laboratories across this region. Samples were tested for E. canis, E. chaffeensis and E. ewingii specific antibodies using peptide microtiter ELISAs.ResultsOverall, E. canis, E. chaffeensis and E. ewingii seroprevalence was 0.8%, 2.8%, and 5.1%, respectively. The highest E. canis seroprevalence (2.3%) was found in a region encompassing Arkansas, Louisiana, Oklahoma, Tennessee and Texas. E. chaffeensis seroreactivity was 6.6% in the central region (Arkansas, Kansas, Missouri, and Oklahoma) and 4.6% in the southeast region (Georgia, Maryland, North Carolina, South Carolina, Tennessee and Virginia). Seroreactivity to E. ewingii was also highest in the central region (14.6%) followed by the southeast region (5.9%). The geospatial pattern derived from E. chaffeensis and E. ewingii seropositive samples was similar to previous reports based on E. chaffeensis seroreactivity in white-tailed deer and the distribution of human monocytic ehrlichiosis (HME) cases reported by the CDC.ConclusionsThe results of this study provide the first large scale regional documentation of exposure to E. canis, E. chaffeensis and E. ewingii in pet dogs, highlighting regional differences in seroprevalence and providing the basis for heightened awareness of these emerging vector-borne pathogens by veterinarians and public health agencies.

Isolation of Tacaribe Virus, a Caribbean Arenavirus, from Host-Seeking Amblyomma americanum Ticks in Florida

Arenaviridae are a family of single stranded RNA viruses of mammals and boid snakes. Twenty-nine distinct mammalian arenaviruses have been identified, many of which cause severe hemorrhagic disease in humans, particularly in parts of sub-Saharan Africa, and in Central and South America. Humans typically become infected with an arenavirus through contact with excreta from infected rodents. Tacaribe virus (TCRV) is an arenavirus that was first isolated from bats and mosquitoes during a rabies surveillance survey conducted in Trinidad from 1956 to 1958. Tacaribe virus is unusual because it has never been associated with a rodent host and since that one time isolation, the virus has not been isolated from any vertebrate or invertebrate hosts. We report the re-isolation of the virus from a pool of 100 host-seeking Amblyomma americanum (lone star ticks) collected in a Florida state park in 2012. TCRV was isolated in two cell lines and its complete genome was sequenced. The tick-derived isolate is nearly identical to the only remaining isolate from Trinidad (TRVL-11573), with 99.6% nucleotide identity across the genome. A quantitative RT-PCR assay was developed to test for viral RNA in host-seeking ticks collected from 3 Florida state parks. Virus RNA was detected in 56/500 (11.2%) of surveyed ticks. As this virus was isolated from ticks that parasitize humans, the ability of the tick to transmit the virus to people should be evaluated. Furthermore, reservoir hosts for the virus need to be identified in order to develop risk assessment models of human infection.

Cultivation of Rickettsia amblyommii in tick cells, prevalence in Florida lone star ticks (Amblyomma americanum)

BackgroundRickettsia amblyommii is a bacterium in the spotted fever group of organisms associated with the lone star tick (LST), Amblyomma americanum. The LST is the most commonly reported tick to parasitize humans in the southeastern US. Within this geographic region, there have been suspected cases of Rocky Mountain spotted fever (RMSF) where the causative agent, R. rickettsii, was not identified in the local tick population. In these areas, patients with clinical signs of RMSF had low or no detectable antibodies to R. rickettsii, resulting in an inability to confirm a diagnosis.MethodsR. amblyommii was cultivated from host-seeking LSTs trapped in Central Florida and propagated in ISE6 (Ixodes scapularis) and AAE2 (A. americanum) cells. Quantitative PCR targeting the 17-kD gene of Rickettsia spp. identified the genus of the organism in culture. Variable regions of gro EL, gtl A and romp A genes were amplified and sequenced to confirm the species. The prevalence of R. amblyommii in LSTs within the geographic region was determined by qPCR followed by conventional PCR and direct sequencing.ResultsAnalyses of amplified sequences from the cultured organism were 100% homologous to R. amblyommii. The overall prevalence of Rickettsia spp. in the local population of LSTs was 57.1% and romp A sequence analysis identified only R. amblyommii in LSTs.ConclusionsA Florida strain of R. amblyommii was successfully cultivated in two tick cell lines. Further evaluation of the new strain and comparisons to the other geographic strains is needed. The prevalence of this SFG organism in the tick population warrants further investigation into the organism’s ability to cause clinical disease in mammalian species.

Prevalence of Tick-Borne Pathogens in Host-Seeking Amblyomma americanum (Acari: Ixodidae) and Odocoileus virginianus (Artiodactyla: Cervidae) in Florida

Abstract Amblyomma americanum (L.), the lone star tick, is an aggressive tick that is expanding its geographic range within the United States. This tick is the vector for the human and veterinary pathogens Ehrlichia chaffeensis and Ehrlichia ewingii and is associated with other microbes of unspecified pathogenicity including Rickettsia amblyommii, Panola Mountain Ehrlichia, and Borrelia lonestari. In Florida, there has been sparse contemporary data on the prevalence of these organisms in host-seeking lone star ticks. To determine the prevalence of this tick and associated microbes in North Central Florida state parks, ∼1,500 lone star tick specimens were collected between 2010 and 2012 analyzed by polymerase chain reaction (PCR) sequencing. Additionally, 393 white-tailed deer, Odocoileus virginianus (Zimmerman), samples were analyzed for pathogen prevalence using molecular methods and serology. In lone star ticks, 14.6, 15.6, and 57.1% were positive for E. chaffeensis, E. ewingii, and Rickettsia spp. DNA, respectively. Panola Mountain Ehrlichia or B. lonestari DNA were each detected in nearly 2% of tick specimens. In white-tailed deer, 7.3% were PCR positive for E. chaffeensis, 6.0% for E. ewingii, and 3.2% for rickettsial species. Approximately 45% of white-tailed deer specimens had antibodies to Ehrlichia spp., and <1% had antibodies to Borrelia burgdorferi. In summary, E. chaffeensis, E. ewingii, and spotted fever group rickettsia are highly prevalent in host-seeking lone star ticks and in white-tailed deer in Florida. The molecular and serological evidence of these microbes underscore their zoonotic potential in this region.

Development of a Quantitative PCR Assay for Differentiating the Agent of Heartwater Disease, Ehrlichia ruminantium, from the Panola Mountain Ehrlichia

Panola Mountain Ehrlichia (PME) is an emerging Ehrlichia sp. reported in ten US states. Based on the sequence homology of all known genes, PME is closely related to Ehrlichia ruminantium (ER), the causative agent of heartwater. Heartwater is an economically important tick-borne disease of cattle, sheep and goats responsible for stock losses in sub-Saharan Africa. Unfortunately, ER was imported to the Caribbean islands in the 19th century, and the presence of this foreign animal disease in the Caribbean poses a threat to the US mainland. If introduced, a heartwater outbreak would cause massive losses of naïve livestock. The serologic assay of choice to diagnose heartwater is cross-reactive with Ehrlichia spp., including PME, as we demonstrate here, which would confound disease surveillance in the event of a heartwater outbreak. The purpose of this study was to develop a diagnostic assay capable of rapidly distinguishing between these pathogens. Using synthetic MAP-1B peptides for ER and PME, we tested the cross-reactivity of this assay using sera from infected livestock. The MAP-1B ELISA cannot distinguish between animals infected with PME and ER. Therefore, a dual-plex Taqman™ qPCR assay targeting the groEL gene of PME and ER was developed and validated. Primers were designed that are conserved among all known strains of ER, allowing for the amplification of strains from the Caribbean and Africa. The assay is highly sensitive (10 copies of DNA) and specific. This assay distinguishes between infection with PME and ER and will be a valuable tool in the event of heartwater outbreak on the US mainland, or for epidemiological studies involving either disease-causing organism.

Borrelia burgdorferi DNA absent, multiple Rickettsia spp. DNA present in ticks collected from a teaching forest in North Central Florida

Tick-borne diseases are an emerging public health threat in the United States. In Florida, there has been public attention directed towards the possibility of locally acquired Borrelia burgdorferi sensu stricto, the causative agent of Lyme disease, in association with the lone star tick. The aim of this study was to determine the prevalence of ticks and the pathogens they carry and potentially transmit, such as B. burgdorferi, in a highly utilized teaching and research forest in North Central Florida. Ticks were collected by dragging and flagging methods over a four month period in early 2014, identified, and tested by PCR for multiple pathogens including Anaplasma, Borrelia, Rickettsia, and Ehrlichia species. During the study period the following ticks were collected: 2506 (96.5%) Amblyomma americanum L., 64 (2.5%) Ixodes scapularis Say, 19 (0.7%) Dermacentor variabilis Say, and 5 (0.2%) Ixodes affinis Neuman. Neither Borrelia spp. (0/846) nor Anaplasma spp. (0/69; Ixodes spp. only) were detected by PCR in any of the ticks tested. However, Rickettsia DNA was present in 53.7% (86/160), 62.5% (40/64), 60.0% (3/5) and 31.6% (6/19) of A. americanum, I. scapularis, I. affinis and D. variabilis, respectively. Furthermore, E. chaffeensis and E. ewingii DNA were detected in 1.3% and 4.4% of adult A. americanum specimens tested, respectively. Although receiving an A. americanum bite is likely in wooded areas in North Central Florida due to the abundance of this tick, the risk of contracting a tick-borne pathogen in this specific area during the spring season appears to be low. The potential for pathogen prevalence to be highly variable exists, even within a single geographical site and longitudinal studies are needed to assess how tick-borne pathogen prevalence is changing over time in North Central Florida.

Development of a rapid, simple, and specific real-time PCR assay for detection of pseudorabies viral DNA in domestic swine herds:

Despite successful eradication of pseudorabies virus (PRV) from the commercial pig industry in the United States in 2004, large populations of feral swine in certain regions act as wildlife reservoirs for the virus. Given the threat of reintroduction of the virus into domestic herds, a rapid, reliable, easily implemented assay is needed for detection of PRV. Although a real-time PCR (rtPCR) assay exists, improvements in rtPCR technology and a greater understanding of the diversity of PRV strains worldwide require an assay that would be easier to implement, more cost effective, and more specific. We developed a single-tube, rapid rtPCR that is capable of detecting 10 copies of PRV glycoprotein B (gB) DNA per 20-µL total volume reaction. The assay did not produce a false-positive in samples known to be negative for the virus. The assay was negative for genetically similar herpesviruses and other porcine viruses. Our assay is a highly specific and sensitive assay that is also highly repeatable and reproducible. The assay should be a useful tool for early detection of PRV in pigs in the case of a suspected introduction or outbreak situation.

Natural History of Plasmodium odocoilei Malaria Infection in Farmed White-Tailed Deer

Malaria parasites of the genus Plasmodium are known to infect a variety of vertebrate hosts, including ungulates (hoofed mammals). A recent study found that up to a quarter of white-tailed deer (Odocoileus virginianus) in North America are infected with the parasite Plasmodium odocoilei. In addition to occupying an important ecological niche, white-tailed deer are popular game animals and deer farming represents a rapidly growing industry. However, the effect of P. odocoilei infection in this ecologically and economically important ungulate species is unknown. Our work is significant because (i) we identified a high prevalence of P. odocoilei in farmed deer and (ii) we found evidence for both cleared and persistent infection, as well as an association with decreased survival of young fawns. ABSTRACT White-tailed deer (Odocoileus virginianus), an ecologically and economically important species, are the most widely distributed large animals in North America. A recent study indicated that up to 25% of all white-tailed deer may be infected with Plasmodium odocoilei, a malaria parasite belonging to the distinct clade of ungulate-infecting Plasmodium spp. Because the clinical impact of P. odocoilei on deer health and survival is unknown, we undertook a retrospective longitudinal study of farmed Floridian O. virginianus fawns. We found that a substantial proportion (21%) of fawns acquire malaria infection during the first 8 months of life. Some animals naturally clear P. odocoilei infection, while other animals remain persistently positive. Importantly, we found that animals that acquire malaria parasites very early in life have poor survival compared to animals that remain uninfected. Our report thus provides the first evidence of a clinically significant impact of malaria infection in young deer. IMPORTANCE Malaria parasites of the genus Plasmodium are known to infect a variety of vertebrate hosts, including ungulates (hoofed mammals). A recent study found that up to a quarter of white-tailed deer (Odocoileus virginianus) in North America are infected with the parasite Plasmodium odocoilei. In addition to occupying an important ecological niche, white-tailed deer are popular game animals and deer farming represents a rapidly growing industry. However, the effect of P. odocoilei infection in this ecologically and economically important ungulate species is unknown. Our work is significant because (i) we identified a high prevalence of P. odocoilei in farmed deer and (ii) we found evidence for both cleared and persistent infection, as well as an association with decreased survival of young fawns.

Macacine Herpesvirus 1 Antibody Prevalence and DNA Shedding among Invasive Rhesus Macaques, Silver Springs State Park, Florida, USA

We compiled records on macacine herpesvirus 1 (McHV-1) seroprevalence and, during 2015–2016, collected saliva and fecal samples from the free-ranging rhesus macaques of Silver Springs State Park, a popular public park in central Florida, USA, to determine viral DNA shedding and perform sequencing. Phylogenetic analysis of the US5 and US5-US6 intragenic sequence from free-ranging and laboratory McHV-1 variants did not reveal genomic differences. In animals captured during 2000–2012, average annual seroprevalence was 25% ± 9 (mean ± SD). We found 4%–14% (95% CI 2%–29%) of macaques passively sampled during the fall 2015 mating season shed McHV-1 DNA orally. We did not observe viral shedding during the spring or summer or from fecal samples. We conclude that these macaques can shed McHV-1, putting humans at risk for exposure to this potentially fatal pathogen. Management plans should be put in place to limit transmission of McHV-1 from these macaques.