A new method for in vitro feeding of Rhipicephalus australis (formerly Rhipicephalus microplus) larvae: a valuable tool for tick vaccine development

Abstract

Background: Rhipicephalus microplus is a hard tick that has a major impact on cattle health in tropical and subtropical regions because it feeds on cattle and is implicated in the transmission of pathogens that cause diseases such as bovine anaplasmosis and babesiosis. Presently, acaricides are used to control tick infestation but this is becoming increasingly less effective due to the emergence of tick strains that are resistant to one or more classes of acaricides. Anti-tick vaccines are a promising alternative to control tick infestation in cattle. The life-cycle and host preference of R. microplus, however, makes vaccine research in cattle costly and would therefore greatly benefit from an in vitro screening system.

Methods: To this aim, a stacked 24-well in vitro feeding system was designed in which the blood meal was administered in a chamber on top of the compartment containing the ticks, exploiting their anti-gravitational tendency. Both compartments were separated by a special feeding membrane, which was made by applying a silicone mixture to a gold beater's skin (baudruche membrane) with a paint roller to create a slightly uneven surface of 17-40 μm variable thickness. To further stimulate feeding, the membrane was treated with bovine hair extract and the unit was placed at 37 °C with 90% RH and 5% CO₂.

Results: Using this set-up with Rhipicephalus australis (formerly Rhipicephalus microplus), a larval engorgement rate of up to 71% could be achieved. The larvae could successfully feed on blood, but also on serum. The latter allows easy screening of the effect of sera that are raised against tick proteins on feeding. As an example, serum from cattle that were vaccinated with the Bm86 midgut protein of R. microplus significantly reduced larval engorgement rates by 42%.

Conclusion: The in vitro feeding system's high throughput design and its ability to measure statistically significant anti-tick effects in sera from immunized cattle enables screening of multiple vaccine candidates in a cost-effective manner.

Keywords: Artificial tick feeding; In vitro screening; Larvae; Rhipicephalus australis; Rhipicephalus microplus.

Fig. 1

In vitro feeding unit. a Schematic representation: The wells of the upper layer contained the test serum or blood (1). The top was covered with an Elisa plate seal or Parafilm to maintain sterility. This layer was separated from the second layer by the feeding membrane that was placed with the silicone side facing downwards (2). The wells of the second layer contained the ticks (3). To contain the ticks, this layer was separated from the third layer with netting (4). The third layer functioned as support and allowed gas exchange between the environment and the tick compartments (5). b A frontal view of the unit. c Top view of the feeding unit, R. australis larvae can be seen through the membrane

Fig. 2

R. australis larvae on the feeding membrane. Most of the larvae have engorged with serum (left) or blood (right). Examples of larvae considered unengorged are indicated with an arrow

Fig. 3

Effect of silicone application method. Percentage of ticks considered to have engorged after 48 h. Silicone application with a paint roller double the number of larvae that have engorged

Fig. 4

Effect of feeding time on percentage of engorged larvae. The number of engorged larvae increased significantly 48 h after feeding. Feeding for 72 h did not increase the total number of larvae that have engorged as compared to feeding for 48 h

Fig. 5

Effect of feeding medium on larval engorgement. Engorgement on defribrinated blood was compared to serum alone. In this experiment two independent feeding units were used for each feeding medium. Fifty-nine percent of the larvae with access to serum engorged compared to the 71% feeding on blood

Fig. 6

Specificity and IgG response against Bm86. Serum reactivity was tested with ELISA. Baculovirus expressed Bm86 was captured with Rabbit IgG raised against Pichia expressed Bm86. The 2log end titer of immune serum is 17

Fig. 7

Effect of Bm86 vaccination on percentage of larvae that have engorged. Vaccination reduced the number of engorged larvae by 42%