Arthur R. Alleman

Expression of Multiple Outer Membrane Protein Sequence Variants from a Single Genomic Locus of Anaplasma phagocytophilum

ABSTRACT Anaplasma phagocytophilum is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of A. phagocytophilum within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism Anaplasma marginale, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of A. phagocytophilum, MSP2(P44), is homologous to MSP2 of A. marginale, has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in A. phagocytophilum. As in A. marginale, the msp2(p44) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain msp2(p44) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in msp2(p44) mRNA. These data suggest that, similarly to A. marginale, A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy.

Analysis of the Anaplasma marginale genome by pulsed-field electrophoresis

Anaplasma marginale is a rickettsial parasite of bovine erythrocytes causing world-wide economic losses in livestock production. Despite its importance, little is known about this rickettsia at a molecular level because it has not been cultured in vitro, and there is no small-animal model. Although several genes have been cloned and sequenced, the gross genome structure of the organism has not yet been well characterized. We separated intact bovine erythrocytes from leucocytes, and determined the genome size of A. marginale by use of restriction endonuclease cleavage and pulsed-field gel electrophoresis (PFGE). A value of 56 mol% G+C was obtained for this genome by spectral analysis. Undigested A. marginale DNA failed to migrate under several different electrophoretic conditions, indicating a circular genome. Digestions of intact A. marginale DNA were performed using restriction endonucleases NotI, SfiI and PacI. Complete digestion with SfiI resulted in 12 distinct bands ranging in size from 14 to 170 kbp. Total size determined by addition of SfiI-digested fragments was approximately 1200 kbp. PacI cleaved the A. marginale genome from three different isolates into just three fragments, of 598, 557 and 97 kbp. Incomplete digestion produced a band measuring 1250 kbp. These results indicate that A. marginale has a circular genome between 1200 and 1260 kbp, with a G+C content of 56 mol%.

Comparative Experimental Infection Study in Dogs with Ehrlichia canis, E. chaffeensis, Anaplasma platys and A. phagocytophilum

Dogs acquire infections with the Anaplasmataceae family pathogens, E. canis, E. chaffeensis, E. ewingii, A. platys and A. phagocytophilum mostly during summer months when ticks are actively feeding on animals. These pathogens are also identified as causing diseases in people. Despite the long history of tick-borne diseases in dogs, much remains to be defined pertaining to the clinical and pathological outcomes of infections with these pathogens. In the current study, we performed experimental infections in dogs with E. canis, E. chaffeensis, A. platys and A. phagocytophilum. Animals were monitored for 42 days to evaluate infection-specific clinical, hematological and pathological differences. All four pathogens caused systemic persistent infections detectible throughout the 6 weeks of infection assessment. Fever was frequently detected in animals infected with E. canis, E. chaffeensis, and A. platys, but not in dogs infected with A. phagocytophilum. Hematological differences were evident in all four infected groups, although significant overlap existed between the groups. A marked reduction in packed cell volume that correlated with reduced erythrocytes and hemoglobin was observed only in E. canis infected animals. A decline in platelet numbers was common with E. canis, A. platys and A. phagocytophilum infections. Histopathological lesions in lung, liver and spleen were observed in all four groups of infected dogs; infection with E. canis had the highest pathological scores, followed by E. chaffeensis, then A. platys and A. phagocytophilum. All four pathogens induced IgG responses starting on day 7 post infection, which was predominantly comprised of IgG2 subclass antibodies. This is the first detailed investigation comparing the infection progression and host responses in dogs after inoculation with four pathogens belonging to the Anaplasmataceae family. The study revealed a significant overlap in clinical, hematological and pathological changes resulting from the infections.

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.

The effects of low‐level laser therapy in a rat model of intestinal ischemia–reperfusion injury

To investigate the effects of low‐level laser therapy applied to the serosal surface of the rat jejunum following ischemia and reperfusion.