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Experimental infection of Rhipicephalus sanguineus with Rickettsia conorii conorii

2009; Elsevier BV; Volume: 15; Linguagem: Inglês

10.1111/j.1469-0691.2008.02259.x

1469-0691

Cristina Socolovschi, Kotaro Matsumoto, Philippe Brouqui, Didier Raoult, Philippe Parola,

Vector-Borne Animal Diseases

Little is known about the relationships between Rickettsia conorii conorii, the agent of Mediterranean spotted fever (MSF), and its main vector, the brown dog tick Rhipicephalus sanguineus [1Parola P Paddock C Raoult D Tick-borne rickettsioses around the world: emerging diseases challenging old concepts.Clin Microbiol Rev. 2005; 18: 719-756Crossref PubMed Scopus (750) Google Scholar]. Matsumoto et al. recently reported a high mortality of Rh. sanguineus group ticks infected with R. conorii conorii by several methods including the use of a bacteraemic rabbit [2Matsumoto K Brouqui P Raoult D Parola P Experimental infection models of ticks of the Rhipicephalus sanguineus group with Rickettsia conorii.Vector Borne Zoonotic Dis. 2005; 5: 363-372Crossref PubMed Scopus (38) Google Scholar]. It had been speculated that reasons for this reduction in fitness might include the geographic origin of the ticks, which came from Thailand where R. conorii conorii has not been reported, or was associated with the pathogen load acquired during laboratory experiments. Here we present complementary experiments to test these hypotheses. We used colonies of uninfected larval and nymphal stages of ticks originating from southern France, morphologically and molecularly (AF150020) identified as Rh. sanguineus, and maintained in our laboratory since 2004. For the blood meal, we placed ticks on rabbits to feed until repletion and engorged ticks were collected, as described previously [2Matsumoto K Brouqui P Raoult D Parola P Experimental infection models of ticks of the Rhipicephalus sanguineus group with Rickettsia conorii.Vector Borne Zoonotic Dis. 2005; 5: 363-372Crossref PubMed Scopus (38) Google Scholar]. Fresh R. conorii conorii suspension was prepared, and titre of the suspension was determined using both Microcyte Biodetect (Oslo, Norway) and plaque assay technique (2 × 106 and 3.3 × 105 bacteria/10 mL PBS), in two successive experiments (Exp.1 and Exp.2) [3Harding CL Lloyd DR McFarlane CM Al-Rubeai M Using the Microcyte flow cytometer to monitor cell number, viability, and apoptosis in mammalian cell culture.Biotechnol Prog. 2000; 16: 800-802Crossref PubMed Scopus (30) Google Scholar]. These concentrations were chosen according to the results of previous experiments [2Matsumoto K Brouqui P Raoult D Parola P Experimental infection models of ticks of the Rhipicephalus sanguineus group with Rickettsia conorii.Vector Borne Zoonotic Dis. 2005; 5: 363-372Crossref PubMed Scopus (38) Google Scholar] (Socolovschi et al., unpublished). The percentage of live bacteria in all suspensions was calculated using Bactlight viability kit (Invitrogen Carlsbad, CA, USA) (85.4 ± 3.8%). The infection of ticks with rickettsiae through a bacteraemic rabbit was performed by direct inoculation (DI), and by continuous inoculation for 2 h (CI) in four serial days via a skin device, to potentially increase the duration of the bacteraemia of rabbits [2Matsumoto K Brouqui P Raoult D Parola P Experimental infection models of ticks of the Rhipicephalus sanguineus group with Rickettsia conorii.Vector Borne Zoonotic Dis. 2005; 5: 363-372Crossref PubMed Scopus (38) Google Scholar]. To monitor the bacteraemia in the rabbit, 500 microl of EDTA and heparin anticoagulated blood samples were collected from the rabbit before inoculation and at 30 min, 1 h, 2 h, 4 h and 6 h after inoculation on each day. Blood with heparin was inoculated into shell vials containing a monolayer of L929 cells as previously described [4Raoult D Roussellier P Vestris G Galicher V Perez R Tamalet J Susceptibility of Rickettsia conorii and R. rickettsii to pefloxacin, in vitro and in ovo.J Antimicrob Chemother. 1987; 19: 303-305Crossref PubMed Scopus (12) Google Scholar]. To test infection of the ticks with rickettsiae, DNA was extracted from: engorged ticks, ticks that died during moulting, and 200 microl of blood collected in EDTA by use of the QIAamp Tissue and Blood kit (Qiagen, Duesseldorf, Germany) according to the manufacturer's directions. These samples were tested by PCR targeting the gltA gene and ompA genes, using primers CS409-Rp1258n and 190.70-190.701, respectively [2Matsumoto K Brouqui P Raoult D Parola P Experimental infection models of ticks of the Rhipicephalus sanguineus group with Rickettsia conorii.Vector Borne Zoonotic Dis. 2005; 5: 363-372Crossref PubMed Scopus (38) Google Scholar]. Relative quantity of R. conorii conorii in blood samples was determined by semi-quantitative PCR using primers, RKNDO3F-RKNDO3R, by the Quantitect probe method [5Svraka S Rolain JM Bechah Y Gatabazi J Raoult D Rickettsia prowazekii and real-time polymerase chain reaction.Emerg Infect Dis. 2006; 12: 428-432Crossref PubMed Scopus (17) Google Scholar]. Four serial logarithmic dilutions of DNA samples of R. montanensis were used to make a standard curve for quantification. DNA samples from R. montanensis, and non-infected ticks and distilled sterile water were used as positive and negative control of PCR, respectively. GraphPad PrismTM v 2.0. (La Jolla, USA) was used to perform the statistical analyses. The injection schedule encompassed the entire duration of the tick blood meal, which was completed after 4 days of attachment for all stages. Rickettsaemia varied in the rabbit depending on the method of inoculation (DI or CI). Peak rickettsaemia was obtained 30 min after DI inoculation, and at 120 min, the end of the perfusion, in model CI. Quantitative PCR only detects the DNA of rickettsiae but not necessarily living bacteria, but shell vial culture proved that R. conorii conorii was alive in rabbit blood as long as 6 h after inoculation. Of 48 engorged nymphs chosen randomly in Exp.1, 36 ticks (75%) in the DI model and 10 ticks (25%) in CI model tested by PCR were positive (p = 0.0001). In Exp.2, one out of the 20 tested nymphs in the DI model and four out of the 20 tested nymphs of CI model were positive (Table 1). The infection rates between Exp.1 and Exp.2 for the DI model were significantly different (p = 0.0001). Mortality of the larvae during moulting in Exp.1 was 53.6% (438/817) for the DI model and 56.8% (261/459) for the CI model. In Exp.1, mortality of nymphs was 93.4% (128/137) in DI and 41.3% (48/116) in CI (p = 0.0001). The mortality of larvae and nymphs in Exp.1 was higher than those in Exp.2 (p = 0.0001). The infection rates of dead nymphal ticks were 85–90%. We tested four pools of five adults in Exp.1, and 20 pools of 20 nymphs and 20 pools of five adults after moulting of each model in Exp.2. Only one pool of five adults of model DI in Exp.2 was positive. No colony of infected ticks was obtained in the subsequent generations of these ticks.TABLE 1Infection rate with R. conorii conorii of larval and nymphal Rhipicephalus sanguineus ticks through a bacteraemic rabbit, mortality during moulting and infection rate of dead ticks during moultingLarvaeNymphsInfection rate before moulting (%)Mortality during moultingInfection rate of nymphs after moultingInfection rate before moulting (%)Mortality during moultingInfection rate of adults after moulting (%)Mortality rate (%)Infection rate of dead larvae (%)Mortality rate (%)Infection rate of dead nymphs (%)Exp.1DI9/50 (18)438/817 (53.6)6/20 (30)NT36/48 (75)128/137 (93.4)19/20 (95)NTCI6/54 (11)261/459 (56.8)2/20 (10)NT10/40 (25)46/116 (41.3)19/20 (95)0/20bFour and 20 pools of five adults were tested.Exp.2DI6/40 (15)40/1246 (3.2)5/20 (25)0/400aTwenty pools of 20 nymphs were tested.1/20 (5)37/337 (11)17/20 (85)1/100bFour and 20 pools of five adults were tested. (1–2)CI8/40 (20)85/1343 (6.3)5/20 (25)0/400aTwenty pools of 20 nymphs were tested.4/20 (20)20/193 (10.4)17/20 (85)0/100bFour and 20 pools of five adults were tested.Exp.1 and Exp.2 p valuep = 0.0001p = 0.0001p = 0.0001Infection rate, PCR positive ticks /PCR tested ticks; mortality rate, number of dead ticks/ number of total ticks.CI, continuous intravenous inoculation; DI, direct intravenous inoculation.a Twenty pools of 20 nymphs were tested.b Four and 20 pools of five adults were tested. Open table in a new tab Infection rate, PCR positive ticks /PCR tested ticks; mortality rate, number of dead ticks/ number of total ticks. CI, continuous intravenous inoculation; DI, direct intravenous inoculation. Our data demonstrate that: (i) the DI model is more effective than the CI model with respect to infecting ticks using a bacteriaemic rabbit; (ii) an inoculum of 2 × 106 R. conorii conorii led to the highest infection rate for the ticks; (iii) the lethal effects of R. conorii conorii on Rh. sanguineus are probably unrelated to the geographical origin of the ticks; and (iv) an optimal rate of infection is accompanied by a great mortality rate of the infected ticks. The mechanisms responsible for rickettsia-induced tick mortality, at least in experimental models, remain unclear and consequences in the natural cycle of Rh. sanguineus and R. conorii are not elucidated. It is possible that the number of rickettsiae in experimental ticks may not correspond to that of R. conorii conorii acquired by ticks in nature. It may be possible that some populations of Rh. sanguineus in natural small foci are better adapted to rickettsial infection. Finally, some rickettsia may have a real negative effect on their tick vectors in nature and are only maintained with the help of animal reservoirs. However, more investigations are needed. Particularly, they should include the study of naturally infected ticks.