Laboratory-InfectedEhrlichia chaffeensisFemale AdultAmblyomma americanumSalivary Glands Reveal Differential Gene Expression

Abstract

Ticks are efficient ectoparasites that are able to steal blood, a rich source of nutrients, from their vertebrate hosts. The nymphal developmental stage of ticks plays an important role for pathogen transmission to human and other animal hosts. In this article, we describe a bloodmeal-based sex differentiation tool to generate adult female ticks infected with Ehrlichia chaffeensis to investigate vector-pathogen interactions (functional genomics and gene expression studies). We demonstrate that there is a correlation between the uptake of blood during nymph attachment and the molting into male or female adult ticks. The data obtained from the bloodmeal experiments suggest that nymphs that molt into females presumably imbibe more blood than those that become male during the nymphal stage. The natural low E. chaffeensis infection rate in female adult Amblyomma americanum (L.) is a major limiting factor to investigate Ehrlichia-Amblyomma interactions. To generate Ehrlichia-infected female adult ticks, we inoculated obligate E. chaffeensis (Arkansas strain) infected DH82 cells into heavier engorged nymphs (> 12 mg) and allowed them to molt. Freshly molted adults were used to test the E. chaffeensis infection rate. E. chaffeensis genomic DNA was extracted from individual unfed and partially blood fed tick midgut and salivary gland tissues. The tissue samples were tested for the presence of E. chaffeensis using the nested polymerase chain reaction process. Polymerase chain reaction-amplified fragments were detected in unfed and partially fed tissues, demonstrating successful E. chaffeensis infection of tick tissues. This method was used to successfully show differential expression of selected tick genes in E. chaffeensis-infected midguts and salivary glands.

Amblyomma americanum ticks infected with in vitro cultured wild-type and mutants of Ehrlichia chaffeensis are competent to produce infection in naïve deer and dogs

“…Goddard (2003) also successfully inoculated A. americanum ticks with a bacterial pathogen, Rickettsia parkeri , but were unable to produce infections in guinea pigs from those ticks. Karim et al (2012) demonstrated that nymphal lone star ticks could be inoculated with culture-grown E. chaffeensis and the infection in ticks was greater than 80% when assessed after molting to the adult stage, although the authors did not describe its validity for the pathogen transmission. Recently, we validated this study in generating E. chaffeensis infected ticks by a needle inoculation method (Cheng et al, 2015).…”

Choice of a Stable Set of Reference Genes for qRT-PCR Analysis in <I>Amblyomma maculatum</I> (Acari: Ixodidae)

“…Before infestation on the host, all unfed ticks were kept at Ϸ28ЊC, with 90% relative humidity (RH), for a photoperiod of 14:10 (L:D) h. Ticks were infested on host and 20 Ð25 ticks were pulled off the host at each time point, 0, 12, 18, 24, 36, 48, 72, 96, 120, 144, 168, and 192 h. Within 4 h of being removed from the sheep, tick tissues were collected from the ticks in ice-cold 100 mM 3-(N-Morpholino)-propanesulfonic acid (MOPS) buffer containing 20 mM ethylene glycol bis-(b-aminoethyl ether)-N, N, NЈ, NЈ-tetraacetic acid (EGTA), pH 6.8. Once dissected, salivary glands and midguts were gently washed in the same ice-cold buffer (Karim et al 2012). Dissected tissues from each time point were pooled together and stored in RNAlater (Ambion, Austin, TX) before extracting total RNA or directly stored in lysis buffer and stored at Ϫ80ЊC.…”

“…Total RNA was puriÞed from pooled salivary glands and midguts from unfed and blood adult female A. maculatum using illustra RNAspin Mini RNA isolation kit (GE Healthcare, Amersham Place, Buckinghamshire, United Kingdom), according to the manufacturerÕs instruction using a slight modiÞcation for tick RNA extractions (Karim et al 2011(Karim et al , 2012. Brießy, the total RNA was eluted into nuclease free water and the concentration of total RNA was determined using the Nanodrop spectrophotometer and stored at Ϫ80ЊC (Karim et al 2011(Karim et al , 2012. The total RNA was reverse transcribed using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase according to manufacturerÕs protocol (Invitrogen).…”

“…Approximately 1,500 ng of total RNA was reverse transcribed using MMLV reverse transcriptase according to manufacturerÕs protocol (Invitrogen). The cDNA used for qRT-PCR gene expression analysis was Ϸ25 ng/l (Karim et al 2012). First strand cDNA was used to measure mRNA levels through qRT-PCR.…”

“…All samples were run in triplicate with NTC controls. The C1000 Thermal Cycler Þtted with a CFX96 Real Time System (Bio-Rad) was used to control the temperature proÞle that was: 10 min at 95ЊC, followed by 35 cycles of 15 s at 95ЊC, 30 s at 60ЊC, and 30 s at 72ЊC with a ßuorescence read at the end of this 30 s step (Karim et al 2012). To conÞrm the ampliÞcation size, a 2% agarose gel electrophoresis of the PCR product was run for each primer pair.…”