Widespread distribution and high prevalence of an alpha-proteobacterial symbiont in the tick Ixodes ricinus.

The tick Ixodes ricinus is responsible for the transmission of a number of bacterial, protozoan and viral diseases to humans and animals in Europe and Northern Africa. Female I. ricinus from England, Switzerland and Italy have been found to harbour an intracellular alpha-proteobacterium, designated IricES1, within the cells of the ovary. IricES1 is the only prokaryote known to exist within the mitochondria of any animal or multicellular organism. To further examine the distribution, prevalence and mode of transmission of IricES1, we performed polymerase chain reaction screening of I. ricinus adults from 12 countries across its geographic distribution, including tick colonies that have been maintained in the laboratory for varying periods of time. IricES1 was detected in 100% of field-collected female ticks from all countries examined (n = 128), while 44% of males were found to be infected (n = 108). Those males that are infected appear to harbour fewer bacteria than females. Sequencing of fragments of the 16S rRNA and gyrB genes revealed very low nucleotide diversity among various populations of IricES1. Transmission of IricES1 from engorged adult females to eggs was found to be 100% (n = 31). In tick colonies that had been maintained in the laboratory for several years, a relatively low prevalence was found in females (32%; n = 25). To our knowledge, IricES1 is the most widespread and highly prevalent of any tick-associated symbiont.

Seroepidemiology of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in wild mice captured in northern Turkey.

An expedition across the Asian part of the Black Sea coast and national parks of Northern Turkey was organized in the summer of 2001 to investigate the presence of Borrelia burgdorferi sensu lato (s.l.), Lyme borreliosis agent, and Anaplasma phagocytophilum, human granulocytic ehrlichiosis, agent, in wild mice. A total of 65 Apodemus flavicollis, Apodemus sylvaticus, Microtus epiroticus, Crocidura suaveolens and Mus macedonicus, were captured. Two out of 22 Apodemus sylvaticus specimens were seropositive for B. afzelii by enzyme-linked immunosorbent assay as confirmed by Western blotting, however cultures of skin and bladder samples from all small mammals in Barbour-Stoenner-Kelly's medium-II remained negative for B. burgdorferi s.l. All sera tested were negative for Anaplasma phagocytophilum by indirect immunofluorescent assay. The prevalence of B. burgdorferi s.l. and Anaplasma phagocytophilum is low in wild mice of the Asian part of Northern Turkey.

Complement evasion by the Lyme disease spirochete Borrelia burgdorferi grown in host-derived tissue co-cultures: role of fibronectin in complement-resistance.

The effectiveness of complement-mediated killing of Borrelia burgdorferi, the causative agent of Lyme disease, in the presence of host-derived tissues was studied. Second and high passage forms of B. burgdorferi 297 isolate were grown in a LEW/N rat joint tissue co-culture system and in artificial BSK medium. Guinea pig complement and third week immune serum from hamsters with experimental Lyme disease were added to the cultures. Both high and low passage borrelia grown in BSK medium died and did not revive after 3 weeks incubation in BSK medium. However, 5-12% of tissue co-cultured borrelia survived the first complement-mediated lysis. Repeated re-growth and lysis cycles in tissue co-culture resulted in isolation of an 85% complement-resistant population of B. burgdorferi. Joint tissue culture supernatant collected on the third day of tissue culture, and fibronectin (25 micrograms/ml), also protected spirochetes from complement-mediated lysis in contrast to BSK or fresh co-culture medium. Complement-mediated lysis may not be an effective mechanism in eradication of borrelia, and the chronicity of Lyme disease may be due to resistance of B. burgdorferi variants to host immune defense mechanisms in the presence of host-derived tissues.

Retention of B. burgdorferi pathogenicity and infectivity after multiple passages in a co-culture system.

In vitro cultivation of B. burgdorferi in BSK medium results in the loss of infectivity and pathogenicity after repeated passages. To prevent this loss, a feeder layer of tibio-tarsal joint tissue derived from newborn LEW/N rats was grown on Cytodex 3 microcarriers in ESG (formerly BSKE), a novel medium developed to support the growth of both the feeder layer and B. burgdorferi. A new pathogenic isolate (FNJ) and a high passage, non-pathogenic strain (TNJ) grew well in this co-culture system with high yields of viable organism. FNJ caused no growth inhibition or visible damage to the cells in the feeder layer. FNJ remained arthritogenic for newborn LEW/N rats after 22 passages in the co-culture system, but lost its arthritogenicity after 7 passages when cultured in BSK medium. This borrelia-mammalian tissue co-culture technique presents an experimental system to study the long term interactions of B. burgdorferi with the infected host tissues in vitro, as well as facilitate diagnostic tests and vaccine development.