Louis A. Magnarelli

Immunization of Mice Against West Nile Virus with Recombinant Envelope Protein

West Nile (WN) virus is a mosquito-borne flavivirus that emerged in the United States in 1999 and can cause fatal encephalitis. Envelope (E) protein cDNA from a WN virus isolate recovered from Culex pipiens in Connecticut was expressed in Escherichia coli. The recombinant E protein was purified and used as Ag in immunoblot assays and immunization experiments. Patients with WN virus infection had Abs that recognized the recombinant E protein. C3H/HeN mice immunized with E protein developed E protein Abs and were protected from infection with WN virus. Passive administration of E protein antisera was also sufficient to afford immunity. E protein is a candidate vaccine to prevent WN virus infection.

Biology of Ticks

Ticks are among the most significant blood-sucking arthropods worldwide. They transmit various pathogens that can cause disease and death in people, domesticated animals, and wildlife. Ticks have several morphologic features and physiologic mechanisms that facilitate host selection, ingestion of vertebrate blood, mating, survival, and reproduction. Although the natural history of ticks varies considerably among species, these arthropods are well-adapted to survive in tropical, temperate, and even subarctic habitats. Key factors, including the reversion of agricultural lands to forests and a close association between people and ticks, have greatly increased the risk of tick bite and human disease.

SPIROCHETES IN TICKS AND ANTIBODIES TO BORRELIA BURGDORFERI IN WHITE-TAILED DEER FROM CONNECTICUT, NEW YORK STATE, AND NORTH CAROLINA

Ticks were screened for spirochetes and serum samples from white-tailed deer (Odocoileus virginianus) were assayed for antibodies to Borrelia burgdorferi during 1983–1984. Using fluorescein isothiocyanate-labeled rabbit antibodies produced to B. burgdorferi, the etiologic agent of Lyme disease, spirochetes were detected in Ixodes dammini (10.5% of 1,193) and Dermacentor albipictus (0.6% of 157) adults from Connecticut, I. dammini nymphs (49.1% of 108) and adults (64.7% of 99) from Armonk, New York, and in I. scapularis (0.4% of 531) and Amblyomma americanum (3.5% of 173) adults from North Carolina. Infected ticks were either seeking hosts or feeding on deer during the summer and fall. Direct fluorescent antibody staining also revealed spirochetes in two larvae of I. scapularis that emerged from eggs deposited by separate females in the laboratory. Using indirect immunofluorescence tests, antibodies to B. burgdorferi were identified in white-tailed deer living in tick-infested areas of all three states. Aside from minor cross-reactivity, there was no serologic evidence of Treponema or Leptospira infections. Ixodes dammini is a primary vector of B. burgdorferi in northeastern United States, but in North Carolina, other ixodid ticks may transmit this spirochete to humans and wildlife.

ANTIBODIES TO MULTIPLE TICK-BORNE PATHOGENS OF BABESIOSIS, EHRLICHIOSIS, AND LYME BORRELIOSIS IN WHITE-FOOTED MICE

Serum samples from Peromyscus leucopus (white-footed mouse), collected in Connecticut (USA) in 1983, 1985, and during 1990 to 1993, were analyzed by an enzyme-linked immunosorbent assay (ELISA) or indirect fluorescent antibody (IFA) staining methods for antibodies to Borrelia burgdorferi (strain 2591), Babesia microti, Ehrlichia chaffeensis (Arkansas strain), and Ehrlichia equi (MRK strain). Of the 294 serum samples tested, 160 (54%) contained immunoglobulins to one or more of these pathogens. There were antibodies to two or more etiologic agents in 77 (48%) of the seropositive mice. Although it was uncommon to detect coexisting antibodies to all four pathogens (n = 5 positive mice), E. chaffeensis-reactive antibodies or immunoglobulins to E. equi were present along with those produced to B. burgdorferi and B. microti in 24 other mice. These rodents carry antibodies to several tick-borne pathogens at numerous sites in Connecticut and may play a role in the epizootiology of ehrlichiosis as well as babesiosis and Lyme borreliosis.

The Early Humoral Response in Human Granulocytic Ehrlichiosis

The early antibody response in patients with human granulocytic ehrlichiosis (HGE) and in mice infected with the HGE agent was characterized by using sera to probe lysates of HL-60 cells infected with HGE organisms. Sera were obtained from 18 patients with HGE, mostly within the first 6 weeks of clinical infection, and from mice infected with the HGE agent for up to 3 weeks. A 44-kDa antigen was reactive with IgG in all 18 patients, and IgG to 40-, 65-, and 80-kDa antigens was present in 6, 8, and 7 patients, respectively. In addition, IgM to 40-, 44-, 65-, and 80-kDa antigens was found in 9, 5, 4, and 4 subjects. Immunoglobulins to antigens ranging between 95 and 125 kDa were detected less frequently. HGE agent-infected C3H/HeJ mice had an antibody response similar to that in humans. Thus, the 40- and 44-kDa proteins of the HGE agent elicit early strong antibody responses during infection. Characterization of the antigens recognized by antibodies during HGE should aid in diagnosis and understanding of the disease.

The Emergence of Another Tickborne Infection in the 12-Town Area around Lyme, Connecticut: Human Granulocytic Ehrlichiosis

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne infection, increasingly recognized in areas in which Lyme disease is endemic, but there are few data on the incidence of HGE. Prospective population-based surveillance was conducted in the 12-town area around Lyme, Connecticut, by means of both active and passive methods, from April through November of 1997, 1998, and 1999. Five hundred thirty-seven residents presenting to their primary care provider with an acute febrile illness suggestive of HGE were identified. Of these, 137 (26%) had laboratory evidence (by indirect fluorescent antibody staining or polymerase chain reaction) of HGE; 89 were confirmed cases, and 48 were probable cases. The incidence of confirmed HGE was 31 cases/100,000 in 1997, 51 cases/100,000 in 1998, and 24 cases/100,000 in 1999. A subset of sera was tested by use of immunoblot assays, and results were in agreement with indirect fluorescent antibody methods for 86% of samples analyzed. Thus, HGE is an important cause of morbidity and is now the second most common tickborne infection in southeastern Connecticut.

Bird-feeding ticks transstadially transmit Borrelia burgdorferi that infect Syrian hamsters.

Bird-feeding Ixodes dammini ticks were documented for the first time to successfully molt and transstadially pass Borrelia burgdorferi spirochetes that were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the type B31 strain. Forty-six of 73 blood-engorged larvae and 50 of 66 fully-fed nymphs, removed from wild-caught birds, successfully molted. Borreliae were isolated from 21 of 78 partially- and fully-fed larvae off birds, including six specimens that molted. Spirochete-positive cultures also were obtained from 35 of 60 partially-and fully-fed nymphs that had fed from birds, including 20 nymphs that molted into adult ticks. Transstadially passed borreliae by bird-feeding larval and nymphal I. dammini were infectious to hamsters, leading us to suggest that these ticks are capable of subsequently transmitting infectious spirochetes to mammals, including humans. An isolate of B. burgdorferi, recovered from a bird-feeding larval Ixodes dentatus, was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the B31 strain. This isolate, unlike another from I. dentatus off a cottontail rabbit (Sylvilagus floridanus), had a protein band with a molecular weight of approximately 31,000 that reacted with murine monoclonal antibodies H3TS and H5332 in western blot analysis. Thus, closely related borreliae are present in both I. dentatus and I. dammini.

Comparative reactivity of human sera to recombinant VlsE and other Borrelia burgdorferi antigens in class-specific enzyme-linked immunosorbent assays for Lyme borreliosis

A comparative study of human sera was conducted to determine which purified preparations of 11 recombinant antigens of Borrelia burgdorferi sensu stricto were diagnostically most important in enzyme-linked immunosorbent assays (ELISAs). To assess sensitivity, 20 serum samples obtained 1-6 weeks after onset of illness from 20 persons who had physician-diagnosed erythema migrans (EM) were tested for IgM and IgG antibodies. In tests for IgM antibody, seropositivity of > or = 25% was recorded when ELISAs had separate preparations of protein (p) 37, p41-G, outer-surface protein (Osp) C, OspE, OspF or VlsE antigens. Sera reacted most frequently (80% positive) with VlsE antigen in analyses for IgG antibodies. When results of both class-specific assays were considered for VlsE, OspC or OspF, 90% of the EM cases were serologically confirmed. Results of specificity testing with a further 59 sera from persons who had syphilis, louse-borne relapsing fever, oral infections, rheumatoid arthritis or human granulocytic ehrlichiosis and 28 normal sera indicated no false positive reactions when VlsE antigen was used in tests for IgM antibody. One of the 11 louse-borne relapsing fever sera cross-reacted with VlsE antigen in tests for IgG antibodies. Minor cross-reactivity also occurred when p37, OspC, OspE or OspF antigens were used. Overall, VlsE was the most suitable antigen for laboratory diagnosis of Lyme borreliosis during the early weeks of B. burgdorferi infection because of its high sensitivity and specificity.

Epizootiology of Lyme disease-causing borreliae☆

Abstract Otto Obermeier first observed helical-shaped bacteria, now called borreliae, in the blood of patients diagnosed with epidemic relapsing fever in 1867. 1 The association of borreliae with arthropods was probably first documented in 1903 with the transmission of Borrelia anserina to chickens following bites by soft-bodied ticks. 2 Thereupon, the transmission of endemic relapsing fever-causing borreliae to humans following bites by soft-bodied ticks was reported. 3 Thus, the serial transfer of borreliae from animals to ticks and then to humans was documented. Although erythema migrans in Europe was recognized to be associated with hard-bodied tick bites, 4 to be caused by a bacterium susceptible to penicillin, 5 and to be transmissible from human to human by skin grafts, 6 its spirochetal etiology remained unknown until Willy Burgdorfer observed borreliae in the hard-bodied tick Ixodes dammini and, with Allan Barbour, cultured the spirochetes. 7 The ability to culture borreliae 8 from tissues taken from ticks, 7 humans, 9,10 and wild animals 11,12 in a cell-free medium enabled scientists in both the New and Old Worlds to assemble considerable knowledge on the ecology of this illness now known as Lyme disease or Lyme borreliosis. In this article, the interactions of the causative agent, Borrelia burgdorferi , 13 with its tick vectors, with animal reservoir hosts, and with human disease are reviewed.

INFECTIONS OF GRANULOCYTIC EHRLICHIAE AND BORRELIA BURGDORFERI IN WHITE-TAILED DEER IN CONNECTICUT

Serum or whole blood samples, obtained from 141 white-tailed deer (Odocoileus virginianus) in Connecticut (USA) during 1980, 1991, and 1996, were analyzed to detect past or current infections of Ehrlichia phagocytophila genogroup organisms and Borrelia burgdorferi. When the BDS or NCH-1 strains of granulocytic ehrlichiae were used separately in indirect fluorescent antibody (IFA) staining methods, antibody positivity rates varied from 25 to 64% in 1991 and 1996, respectively. All 50 sera tested from 1980 collections were negative. Although percentages of sera with B. burgdorferi antibodies, as detected by an enzyme-linked immuno-sorbent assay, also differed (23 to 53%), there were coexisting antibodies to both bacteria in 20 (49%) of 41 sera. In tests on specificity, 19 deer sera with ehrlichial antibodies also were tested by IFA staining procedures for Anaplasma marginale antibodies; one serum with a titer of 1: 5,120 to ehrlichial antigen reacted to A. marginale antigen at a serum dilution of 1:320. In parallel analyses of 69 sera, results of Western blot analyses for ehrlichial infections in deer were concordant (72% agreement) with those of IFA staining methods containing ehrlichial antigen. All positive immunoblots showed bands to peptides of the NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent having molecular masses of about 44, 105, or 110 kDa. In polymerase chain reaction (PCR) studies of blood samples from 63 deer, 11(18%) specimens were positive for 16S ribosomal DNA of an Ehrlichia phagocytophila genogroup organism, whereas 23 (37%) samples were positive for the DNA of the 44 kDa gene of the HGE agent. White-tailed deer are exposed to different tick-borne bacteria in areas where Ixodes scapularis ticks are abundant and may, in some instances, have had concurrent infections.