Characterization of Transstadial Transmission of Rickettsia Amblyommatis in Haemaphysalis Longicornis Using Optimized Artificial Membrane Feeding System

Abstract

Ticks are blood-feeding arthropods and serve as vectors and reservoirs for diverse pathogens. Recent environmental changes have triggered the invasion of ticks into new geographical areas, prompting a public health alert for increased risk of tick-borne diseases. Amblyomma (A.) americanum (lone star tick) has emerged as the most common human-biting tick species in the eastern United States. Amblyomma americanum transmits multiple pathogens, including Rickettsia (R.) amblyommatis, the suspected cause of mild spotted fever rickettsiosis. As an invasive tick species, Haemaphysalis (H.) longicornis (longhorned tick) has rapidly invaded and expanded to high densities in the eastern United States. Haemaphysalis longicornis and A. americanum often share the habitat with preferential feeding on mid- to large-sized animals, such as white-tailed deer. This sympatric association of H. longicornis with A. americanum raises the potential for H. longicornis to acquire pathogens native to A. americanum during blood-feeding on the same host. In its native ranges, H. longicornis transmits R. japonica and R. heilongjiangensis. However, it remains unclear whether H. longicornis can transmit R. amblyommatis abundantly present in A. americanum in the United States. Using artificial membrane feeding, we establish that R. amblyommatis can stably colonize H. longicornis without altering feeding and molting behaviors. Transovarial transmission of R. amblyommatis did not occur in parthenogenetic H. longicornis. However, R. amblyommatis successfully invaded the midgut and salivary glands of H. longicornis, key organ tissues of rickettsial replication and horizontal transmission. Our results suggest that H. longicornis may serve as a vector, but not as a reservoir, for R. amblyommatis transmission.

Figure 1.

The artificial membrane feeding system supports the blood-feeding of Haemaphysalis (H.) longicornis ticks in all growth stages. (A) Photographic images of the assembled six-well plate (inset) and feeding chamber. Representative images of (B) adults and nymphs (repleted versus non-fed) recovered from the feeding experiments, (C) an engorged nymph and a freshly molted adult with exuviae, and (D) a fully engorged adult with eggs.

Figure 2.

Rickettsia (R.) amblyommatis is transstadially transmitted from nymphs and colonizes the salivary glands and midgut of Haemaphysalis (H.) longicornis adults. Cohorts of H. longicornis nymphs ingested sheep blood supplemented with R. amblyommatis or SPG buffer (Mock) through the artificial membrane feeding system. After repletion, engorged nymphal ticks molted into adults. Freshly molted adult ticks were examined to determine the presence of R. amblyommatis in organ tissues by (A and B) polymerase chain reaction (PCR) or (C) immunofluorescent microscopic analyses. Red = actin; green = Rickettsia (white arrows); blue = nucleus.

Figure 3.

Rickettsia (R.) amblyommatis infects distal organ tissues but fails to colonize ovaries in adult Haemaphysalis (H.) longicornis. (A) Engorged H. longicornis ticks were dissected and polymerase chain reaction (PCR)-tested for R. amblyommatis on day 5 or 14, post-feeding. (B) Furthermore, ovaries from adult ticks were examined for R. amblyommatis by PCR.

Figure 4.

Rickettsia (R.) amblyommatis is transstadially transmitted from larvae and persists in Haemaphysalis (H.) longicornis nymphs. H. longicornis larvae were placed in the artificial membrane feeding system (AFS) to feed sheep blood supplemented with R. amblyommatis or SPG buffer (Mock). After repletion, engorged larval ticks molted into nymphs. Freshly molted nymphal H. longicornis ticks were polymerase chain reaction (PCR)-tested for R. amblyommatis.