A comparison of Chryseobacterium indologenes pathogenicity to the soft tick Ornithodoros moubata and hard tick Ixodes ricinus

Abstract

A yellow-pigmented Gram-negative bacterium, Chryseobacterium indologenes, was found in the gut contents of about 65% of soft ticks Ornithodoros moubata from a perishing laboratory colony. The isolated putative pathogen, C. indologenes, was susceptible to cotrimoxazol and addition of this antibiotic (Biseptol 480) to the blood meal significantly decreased the tick mortality rate. The artificial infection of healthy O. moubata by membrane feeding on blood contaminated with C. indologenes was lethal to all ticks at concentrations ⩾10⁶ bacteria/ml. On the contrary, a similar infection dose applied to the hard tick Ixodes ricinus by capillary feeding did not cause significant mortality. Examination of guts dissected from infected O. moubata and I. ricinus revealed that C. indologenes was exponentially multiplied in the soft tick but were completely cleared from the gut of the hard ticks within 1 day. In both tick species, C. indologenes were found to penetrate from the gut into the hemocoel. The phagocytic activity of hemocytes from both tick species was tested by intrahaemocoelic microinjection of C. indologenes and evaluated by indirect fluorescent microscopy using antibodies raised against whole bacteria. Hemocytes from both tick species displayed significant phagocytic activity against C. indologenes. All O. moubata injected with C. indologenes died within 3 days, whereas the increase of the mortality rate of I. ricinus was insignificant. Our results indicate that hard ticks possess much more efficient defense system against infection with C. indologenes than the soft ticks. Thus, C. indologenes infection has the potential to be a relevant comparative model for the study of tick immune reactions to transmitted pathogens.

Introduction

The research on tick pathogens, parasitoids or predators, which may be used as a reasonable alternative to chemicals acaricides in the regulation of tick population, lags far behind the use of a similar strategy for the biological control of plant pests (Samish and Řeháček, 1999). Moreover, better understanding of how pathogens avoid the innate defense mechanisms in the tick can markedly improve knowledge about tick–pathogen interaction in general.

The soft tick, Ornithodoros moubata (Murray, 1877), is a serious disease vector since it transmits the spirochetes Borrelia duttonii, causing African tick-borne relapsing fever in man (Varma, 1956), and the African swine fever virus, having a fatal impact on the raising of domestic pigs in tropical areas of sub-Saharan Africa (Wardley et al., 1983). Ornithodoros moubata has also been recently shown to be a reservoir and vector for the West-Nile virus (Lawrie et al., 2004). Noda et al. (1997) described two groups of Francisella-like endosymbionts in O. moubata, originally identified as Francisella tularensis and Wolbachia persica. The latter bacterium has been later re-classified as Francisella arboreus by Scoles (2004). In the last decade, O. moubata has become a useful laboratory model because of its size and the relative simplicity of artificial feeding using a ParafilmM^® membrane (Schwan et al., 1991). However, artificially fed ticks have the permanent risk of being infected with pathogens that are rarely if ever met in nature (Osborne and Mellor, 1986). Earlier, we have described the mortality of O. moubata ticks caused by rather rare yeast, Candida haemulonii (Loosová et al., 2001). In this work, we describe a much more serious infection of a soft tick colony caused by the Gram-negative Chryseobacterium indologenes. This bacterium was described as Flavobacterium indologenes by Yabuuchi et al. (1983) and then placed in a new genus, Chryseobacterium, by Vandamme et al. (1994). It is widely distributed in soil and aquatic environments, raw meat, and diary sources (Hugo et al., 1999). Although C. indologenes is a rare pathogen of humans, 36 cases of nosocomial infection were reported to be caused by this bacterium, five of which were lethal. It appears to be an emerging problem in Taiwan because of its multi-resistance to antibiotics (Hsueh et al., 1997). Among invertebrates, C. indologenes (tentatively identified as a Flavobacterium sp.) was already reported to be present as a rare symbiont in the gut of the Australian termite Mastotermes darwiniensis (Eutick et al., 1978). It has been recently found to be abundant in the gut of the American cockroach Periplaneta americana, likely as a symbiont (Dugas et al., 2001). A pilot screen based on 16S rDNA sequences discovered the presence of Chryseobacterium sp. among the microbial community inhabiting the midgut of the mosquito Culicoides variipennis (Campbell et al., 2004). Recently, Montasser (2005) reported the isolation of Chryseobacterium meningospeticum from the chicken tick, Argas persicus.

Here, we show that C. indologenes is pathogenic to the soft tick O. moubata, but apparently non-pathogenic to the hard tick I. ricinus. The defense mechanisms against this bacterium in the gut and hemolymph of both tick species were compared.