The Rhipicephalus (Boophilus) microplus Bm86 gene plays a critical role in the fitness of ticks fed on cattle during acute Babesia bovis infection

Abstract

Background: Rhipicephalus (Boophilus) microplus is an economically important tick of cattle involved in the transmission of Babesia bovis, the etiological agent of bovine babesiosis. Commercial anti-tick vaccines based on the R. microplus Bm86 glycoprotein have shown some effect in controlling tick infestation; however their efficacy as a stand-alone solution for tick control has been questioned. Understanding the role of the Bm86 gene product in tick biology is critical to identifying additional methods to utilize Bm86 to reduce R. microplus infestation and babesia transmission. Additionally, the role played by Bm86 in R. microplus fitness during B. bovis infection is unknown.

Results: Here we describe in two independent experiments that RNA interference-mediated silencing of Bm86 decreased the fitness of R. microplus females fed on cattle during acute B. bovis infection. Notably, Bm86 silencing decreased the number and survival of engorged females, and decreased the weight of egg masses. However, gene silencing had no significant effect on the efficiency of transovarial transmission of B. bovis from surviving female ticks to their larval offspring. The results also show that Bm86 is expressed, in addition to gut cells, in larvae, nymphs, adult males and ovaries of partially engorged adult R. microplus females, and its expression was significantly down-regulated in ovaries of ticks fed on B. bovis-infected cattle.

Conclusion: The R. microplus Bm86 gene plays a critical role during tick feeding and after repletion during blood digestion in ticks fed on cattle during acute B. bovis infection. Therefore, the data indirectly support the rationale for using Bm86-based vaccines, perhaps in combination with acaricides, to control tick infestation particularly in B. bovis endemic areas.

Figure 1

Expression of the Bm86 gene in unfed larvae, engorged nymphs, unfed males, and gut and ovaries of R. microplus females. Six biological replicates of larvae (approximately 100 larvae per sample), engorged nymphs (10 nymphs per sample), unfed males (10 males per sample), and individual ovaries and gut of partially engorged females (at day 5 of feeding) were analyzed by reverse transcriptase quantitative real-time PCR. The transcription level of Bm86 was calculate as relative quantity using the delta Ct comparative method normalized by the total amount of RNA used to generate the cDNA. Different letters (a, b) above the bars indicate significant statistical differences (one-way ANOVA, F = 11.456, Tukey post hoc test, P < 0.0001).

Figure 2

Effect of B. bovis infection on the expression of the R. microplus Bm86 gene. At day 5 of feeding, 6 biological replicates of partially engorged female ticks fed on either B. bovis-infected or uninfected cattle were dissected and gene expression was evaluated in individual guts and ovaries by reverse transcriptase quantitative real-time PCR. The transcription level of Bm86 was calculate as relative quantity using the delta Ct comparative method normalized by the total amount of RNA used to generate the cDNA. Different letters (a, b) above the bars indicate significant statistical differences (t test, P < 0.05).

Figure 3

Level of Bm86 transcript in partially engorged R. microplus females injected with Bm86 dsRNA (grey bar) or buffer control (black bar). At day 5 after injection, 6 biological replicates of partially engorged female ticks fed on cattle during acute B. bovis infection were dissected and gene expression was evaluated in individual guts and ovaries by reverse transcriptase quantitative real-time PCR. The presented data is representative of ticks from experiment one however, similar levels of gene silenced were obtained in ticks from experiment two. The transcription level of Bm86 was calculate as relative quantity using the delta Ct comparative method normalized by the total amount of RNA used to generate the cDNA. Bm86 expression was reduced 92.9% (± 9.3%) and 93.8% (± 2.8%) in gut and ovaries, respectively. Different letters (a, b) above the bars indicate significant statistical differences (t test, P < 0.05).

Figure 4

Results of temperature, packed cell volume (PCV) and Babesia bovis parasitemia of calves used to feed the Bm86 silenced ticks and control ticks. Panel A and B show the experimental conditions of experiment one and two, respectively. The upper right chart in the panels A and B show the results of parasitemia of B. bovis in peripheral blood of calves of experiment one and two, respectively. Arrows highlight the difference between experiment one and two and indicate the beginning (arrows down) and end (arrows up) of tick feeding period.

Figure 5

Histological analysis of R. microplus gut sections stained with hematoxylin and eosin and visualized under light microscopy. A representative gut section of a Bm86 silenced tick is shown in panels A and B (magnification of 40× and 63×, respectively) and a representative gut section from a control tick is shown in panels C and D (magnification of 40× and 63×, respectively). The scale bars in the lower right corner of the upper panels represent 50 μm whereas the scale bars in the lower right corner of the lower panels represent 20 μm. The rectangles in the upper panels correspond to the area shown at 63× magnification in the lower panels. The letter "L" indicates the gut lumen and the arrows show the presence of oval-shape corpuscles resembling undigested red blood cells in the apical gut cells of the Bm86 silenced tick.

Figure 6

Kaplan-Meier curves showing the percentage of survival of Bm86 silenced and control R. microplus engorged females after feeding to repletion on cattle during acute B. bovis infection. Panels A and B show the percentage of survival of female ticks of the experiment one and two, respectively. In experiment one, 100% (n = 83) of the replete females of the control group survived the evaluation period whereas 91.6% (33 out of 36) of the replete females survived in the silenced group. In the experiment two, 100% (n = 8) of the replete females of the control group survived the evaluation period whereas only 27.5% (8 out of 29) of the replete females survived in the silenced group. Chi-squared test showed significant differences (P < 0.05) between the control and silenced groups in both experiments.