Fig 1.
Diagram of an artificial membrane feeding system used to feed R. sanguineus larvae and nymphs.
The artificial feeder consists of a glass feeder, animal skin, and tick container. The glass feeder is connected to a 37°C water circulation system to mimic host body temperature.
Fig 2.
Duration of tick feeding experiment of R. sanguineus larvae into nymphs.
Larvae were fed for 5 days and were allowed to complete their molting period, which ranged from 15–45 days. After they become nymphs, they were starved around one month and then fed for 4 days. B. henselae were detected by PCR assay from larval samples (at the end of larval feeding), nymphal samples (after molting completed) and blood samples (daily collected during the nymphal feeding). After 4 days of nymphal feeding, blood from each feeder was collected for Bartonella isolation.
Fig 3.
R. sanguineus larvae and nymphs engorged using goat blood infected or not with B. henselae.
(A) Larvae attached on the mice skin. (B) Engorged larvae detached from the mice skin after 3 days. (C) Engorged larva (left) and semi-engorged larva (right). (D) Semi-engorged nymph (left) and unfed nymph (right).
Table 1.
Attachment rates of R. sanguineus larvae and nymphs fed on mice skin by artificial membrane feeding system.
Fig 4.
B. henselae DNA detection in R. sanguineus larvae and nymphs.
Detection of B. henselae DNA by nested-PCR in representative samples: M, DNA marker; EL1 and EL2, pooled engorged larvae (Experimental group); SEL, pooled semi-engorged larvae (Experimental group); LF, pooled larval feces (Experimental group); N, pooled unfed nymphs (Experimental group); NF, pooled nymphal feces (Experimental group); EL-, pooled engorged larvae (Control group); SEL-, pooled semi-engorged larvae (Control group); LF-, pooled larval feces (Control group); N-, pooled unfed nymphs (Control group); NF-, pooled nymphal feces (Control group); +, positive control (B. henselae DNA); -, negative control (distilled water).
Table 2.
Detection of B. henselae DNA in R. sanguineus larvae engorged on infected blood and in nymphs after molting and maximum likelihood estimation (MLE) of tick infection.
Fig 5.
Detection by nested-PCR of B. henselae DNA in non-infected blood after feeding of nymphs infected at the larval stage.
M, DNA marker; B1—B4, blood samples collected from day 1 to 4, respectively (Experimental group); B1- - B4-, blood samples collected from day 1 to 4, respectively (Control group); +, positive control (B. henselae DNA); -, negative control (distilled water).