Analysis of Babesia bovis infection-induced gene expression changes in larvae from the cattle tick, Rhipicephalus (Boophilus) microplus

Abstract

BackgroundCattle babesiosis is a tick-borne disease of cattle that has severe economic impact on cattle producers throughout the world’s tropical and subtropical countries. The most severe form of the disease is caused by the apicomplexan, Babesia bovis, and transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus, with the most prevalent species being Rhipicephalus (Boophilus) microplus. We studied the reaction of the R. microplus larval transcriptome in response to infection by B. bovis.MethodsTotal RNA was isolated for both uninfected and Babesia bovis-infected larval samples. Subtracted libraries were prepared by subtracting the B. bovis-infected material with the uninfected material, thus enriching for expressed genes in the B. bovis-infected sample. Expressed sequence tags from the subtracted library were generated, assembled, and sequenced. To complement the subtracted library method, differential transcript expression between samples was also measured using custom high-density microarrays. The microarray probes were fabricated using oligonucleotides derived from the Bmi Gene Index database (Version 2). Array results were verified for three target genes by real-time PCR.ResultsTicks were allowed to feed on a B. bovis-infected splenectomized calf and on an uninfected control calf. RNA was purified in duplicate from whole larvae and subtracted cDNA libraries were synthesized from Babesia-infected larval RNA, subtracting with the corresponding uninfected larval RNA. One thousand ESTs were sequenced from the larval library and the transcripts were annotated. We used a R. microplus microarray designed from a R. microplus gene index, BmiGI Version 2, to look for changes in gene expression that were associated with infection of R. microplus larvae. We found 24 transcripts were expressed at a statistically significant higher level in ticks feeding upon a B. bovis-infected calf contrasted to ticks feeding on an uninfected calf. Six transcripts were expressed at a statistically significant lower level in ticks feeding upon a B. bovis-infected calf contrasted to ticks feeding on an uninfected calf.ConclusionOur experimental approaches yielded specific differential gene expression associated with the infection of R. microplus by B. bovis. Overall, an unexpectedly low number of transcripts were found to be differentially expressed in response to B. bovis infection. Although the BmiGI Version 2 gene index (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=b_microplus) was a useful database to help assign putative function to some transcripts, a majority of the differentially expressed transcripts did not have annotation that was useful for assignment of function and specialized bioinformatic approaches were necessary to increase the information from these transcriptome experiments.

Tick-Pathogen Interactions and Vector Competence: Identification of Molecular Drivers for Tick-Borne Diseases

“…Genes involved in immunity, stress, and defense responses showed up-regulation in response to B. bovis infection (Heekin et al, 2012), while genes encoding for calreticulin, kunitz-type serine protease inhibitors and microplusin which exhibits antimicrobial activity, were differentially expressed in B. bovis/B. bigemina infected Rhipicephalus ticks (Rachinsky et al, 2007; Antunes et al, 2012; Heekin et al, 2013; Lu et al, 2016).…”

Interaction of the tick immune system with transmitted pathogens

“…Although genomic sequences of Babesia and tick are available (Pagel Van Zee et al, 2007; Cornillot et al, 2012) and several projects have identified tick genes differently expressed upon Babesia infection (Rachinsky et al, 2007, 2008; Antunes et al, 2012; Heekin et al, 2012), only few tick genes have been shown to be directly implicated in the vector-pathogen interaction (Figure 4 and Table 3). First of them, called longicin (Tsuji and Fujisaki, 2007), is defensin-like protein of H. longicornis exerting anti-microbial and anti-fungal activity.…”

Widespread movement of invasive cattle fever ticks (Rhipicephalus microplus) in southern Texas leads to shared local infestations on cattle and deer

“…This tick species is a major problem for livestock production worldwide because it is the biological vector for disease agents causing bovine babesiosis ( Babesia bovis , B. bigemina ) and anaplasmosis ( Anaplasma marginale ) [2]. Along with the closely related cattle tick ( R. annulatus ) it was likely introduced to the New World by Spanish colonialists.…”