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

Authors: Heekin, Andrew; Guerrero, Felicito; Bendele, Kylie; SALDIVAR, L - University Of Texas; Scoles, Glen; GONDRO, C - University Of New England; NENE, V - International Livestock Research Institute (ILRI) - Kenya; DJIKENG, A - International Livestock Research Institute (ILRI) - Kenya; BRAYTON, K - Washington State University

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/4/2012
Publication Date: 8/7/2012
Citation: Heekin, A.M., Guerrero, F., Bendele, K.G., Saldivar, L., Scoles, G.A., Gondro, C., Nene, V., Djikeng, A., Brayton, K.A. 2012. Analysis of Babesia bovis-induced gene expression changes in the cattle tick, Rhipicephalus (Boophilus) microplus. Parasites & Vectors. 5:162.

Interpretive Summary: Cattle babesiosis is a tick-borne disease of cattle which 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 microbe, 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 used several approaches to study the reaction of the R.microplus transcriptome in response to infection by B. bovis. Ticks 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, dissected adult female gut, and dissected adult female ovary from both the infected and uninfected samples. Subtracted cDNA libraries were synthesized from nucleic acids purified from Babesia-infected gut, ovary and larvae, subtracting with the corresponding uninfected tissues. One thousand DNAs were sequenced from the larval library and 4, 000 DNAs sequenced from the gut and ovary subtracted libraries. These sequences were assembled into a DNA database for assignment of gene function by bioinformatic analysis. Cattle tick microarray experiments were performed to identify genes which are differentially regulated in response to cattle tick infection with B. bovis. These results showed a total of 33, 15, and 24 transcripts were expressed in gut, ovary and larval tissues, respectively, at a statistically significant higher level in ticks feeding upon a B. bovis infected calf compared to ticks feeding on an uninfected calf. A total of 43, 0, and 6 transcripts were expressed in gut, ovary and larval tissues, respectively, at a statistically significant lower level in ticks feeding upon a B. bovis-infected calf compared to ticks feeding on an uninfected calf. We used a similar procedure called serial analysis of gene expression to discover 41 ovarial transcripts whose expression was differentially expressed when feeding upon a B. bovis infected falf compared to ticks feeding on and uninfected calf. All of our experimental approaches yielded specific differential gene expression associated with other infection for. microplus by B. bovis. Overall, a surprisingly low number of transcripts were found to be differentially expressed in response to B. bovis infection. A cattle tick gene database called BmiGI version 2 was useful to help assign putative function to specific transcripts. However, over 50% of the differentially expressed transcripts in the microarray experiments and over 70% of the 10-fold differentially regulated transcripts form the SAGE experiment did not have annotation that was useful for assignment of function.

Technical Abstract: Background: Cattle 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. Results: Ticks 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. Conclusion: Our 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/cgi53 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.