Isolation and Propagation of Laboratory Strains and a Novel Flea-Derived Field Strain of Wolbachia in Tick Cell Lines

Abstract

<jats:p>Wolbachia are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although Wolbachia DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not Wolbachia can infect and grow in tick cells, cell lines from three tick species, Ixodes scapularis, Ixodes ricinus and Rhipicephalus microplus, were inoculated with Wolbachia strains wStri and wAlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an I. scapularis cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of Wolbachia genes. The wStri strain infected Ixodes spp. cells and was maintained through 29 passages. The wAlbB strain successfully infected Ixodes spp. and R. microplus cells and was maintained through 2–5 passages. A novel strain of Wolbachia belonging to the supergroup F, designated wCfeF, was isolated in I. scapularis cells from a pool of Ctenocephalides sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a Wolbachia strain from a flea and the first isolation of any Wolbachia strain outside the “pandemic” A and B supergroups. The study demonstrates that tick cells can host multiple Wolbachia strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of Wolbachia.</jats:p>

Bacterial Pathogens and Symbionts Harboured by Ixodes ricinus Ticks Parasitising Red Squirrels in the United Kingdom

“…While a recent study demonstrated that several strains of Wolbachia can infect and grow in Ixodes spp. tick cells in vitro [ 47 ], this does not prove that ticks themselves carry the bacterium in vivo. Most of the above-mentioned studies on whole ticks did not include a screen for insect or helminth DNA; of those that did, both found a strong correlation between presence of Wolbachia and presence of the wasp I. hookeri [ 44 , 45 ].…”

“…On the other hand, amongst the single ticks on Brownsea Island that were positive for Borrelia spp., the incidence of Borrelia DNA was over twice as high, at 21.3% in ticks that were also positive for Ixodiphagus / Wolbachia DNA (10/47), than the 10.8% infection rate in ticks that were negative for Ixodiphagus / Wolbachia DNA (8/74). As there is no evidence that Wolbachia naturally infects ticks, but is rather present within parasitic insects or nematodes infesting ticks [ 45 , 47 ], any effect on co-infecting pathogenic bacteria is less likely to result from the Wolbachia than directly from the parasite competing for nutrients or interfering with innate immune responses of the tick host. Infestation of I. ricinus nymphs with I. hookeri in The Netherlands was found to be positively correlated with presence of A. phagocytophilum and negatively correlated with presence of B. afzelii and Ca .…”

“…Two negative controls (one for extraction and one with water instead of DNA) were included in the PCR assays along with appropriate positive control DNA as follows: B. burgdorferi s.s. strain KS20 (kindly provided by Prof. Brian Stevenson, University of Kentucky, Lexington, KT, USA) or Borrelia spielmanii (kindly provided by Dr Volker Fingerle, German National Reference Centre for Borrelia , Oberschleißheim, Bavaria, Germany); A. phagocytophilum strain Webster (kindly provided by Prof Didier Raoult, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, Marseille, France and Prof Stephen Dumler, The Johns Hopkins Hospital, Baltimore, MR, USA) or strain Feral Goat grown in tick cells [ 71 ], Ehrlichia ruminantium strain Ball3 grown in tick cells [ 72 ], Rickettsia amblyommatis [ 73 ]; Spiroplasma sp. strain Bratislava1 [ 74 ] or strain DMAR11 [ 16 ], Wolbachia strain wAlbB grown in mosquito or tick cells [ 47 ], and extract of eggs and larvae of I. hookeri (obtained in the present study).…”

Diminutive, degraded but dissimilar: Wolbachia genomes from filarial nematodes do not conform to a single paradigm

“…The first appearance of the 'supergroups' designation dates to 1998 [54], but the concept was popularized later by Lo et al [55]. Most of the molecular characterizations of Wolbachia strains have been based on either single gene or multi-locus phylogenies [53,[55][56][57][58][59][60][61][62][63][64][65]. The supergroups A, B, E, H, I, K, M, N, O, P, Q and S are exclusively composed of symbionts of arthropods [55,57,59,63,[66][67][68][69][70].…”

“…[55]. Most of the molecular characterizations of Wolbachia strains have been based on either single gene or multi-locus phylogenies [53, 55–65]. The supergroups A, B, E, H, I, K, M, N, O, P, Q and S are exclusively composed of symbionts of arthropods [55, 57, 59, 63, 66–70].…”