The widely distributed hard tick, Haemaphysalis longicornis, can retain canine parvovirus, but not be infected in laboratory condition

Abstract

Ticks are known to transmit various pathogens, radically threatening humans and animals. Despite the close contact between ticks and viruses, our understanding on their interaction and biology is still lacking. The aim of this study was to experimentally assess the interaction between canine parvovirus (CPV) and a widely distributed hard tick, Haemaphysalis longicornis, in laboratory condition. After inoculation of CPV into the hemocoel of the ticks, polymerase chain reaction assay revealed that CPV persisted in inoculated unfed adult female ticks for 28 days. Canine parvovirus was recovered from the inoculated ticks using a cell culture, indicating that the virus retained intact in the ticks after inoculation, but significant positive reaction indicating virus infection was not detected in the tick organs by immunofluorescence antibody test using a monoclonal antibody. In the case of ticks inoculated with feline leukemia virus, the virus had shorter persistence in the ticks compared to CPV. These findings provide significant important information on the characteristic interaction of tick with non-tick-borne virus.

Fig. 1.

Detection of CPV gene from tick samples of CPV-inoculated unfed adult ticks. (A) Detection of amplified PCR products of CPV from CPV-inoculated unfed adult ticks, (B) from different organs, and (C) from the hemocytes and the hemolymph of CPV-inoculated unfed adult ticks. The numbers indicate each dpi, and each dpi consists of 3 individual tick samples. N, negative control ticks; P, undiluted CPV stock solution; SG, salivary glands; MG, midgut; FB, fat body; OV, ovary; SY, synganglion; MT, Malpighian tubules; HC, hemocytes; HL, hemolymph.

Fig. 2.

Recovery of CPV from CPV-inoculated unfed adult ticks on 28 dpi was confirmed by fcwf-4 cells (A) and by PCR (B). (A) Intranuclear inclusion bodies (arrowheads) were detected at the second passage of the cell culture. Scale bars indicate 50 µm. (B) Detection of CPV gene from supernatants of the cell culture by PCR. The numbers indicate each passage level of cell culturing. N, cells inoculated with the solution of a pooled sample of 3 homogenized negative control ticks; P, cells inoculated with undiluted CPV stock solution.

Fig. 3.

Detection of CPV antigens from tick organs of 4-day fed adult ticks through IFAT. The sections were incubated with MAb 2D9 followed by goat anti-mouse IgG conjugated with Alexa Fluor 488 (Alexa 488 sections), and nuclei were visualized using DAPI (DAPI sections). Each picture of the organs shows the representative section of the observed. Scale bars indicate 100 µm.

Fig. 4.

Assessment on transstadial and transovarial transmission of CPV in the ticks by PCR. (A) Detection of amplified PCR products from engorged nymphs and newly emerged adult ticks after inoculation with CPV on day 1 post-their last engorgement. The numbers indicate each day post-engorgement, and each day consists of 4 CPV-inoculated and 1 control ticks. Molting was completed between days 10 and 20 post-engorgement. (B) Detection of amplified PCR products from the eggs and larvae originated from CPV-inoculated engorged adult ticks. Three tested and 2 control samples are shown as representatives. The tick L23 gene was amplified as a loading control. N, samples obtained from negative control ticks; CPV, samples obtained from CPV-inoculated ticks; P, undiluted CPV stock solution.

Fig. 5.

Detection of FeLV gene from the FeLV-inoculated unfed adult ticks by RT-PCR. The tick actin mRNA was detected and amplified as a loading control. The numbers indicate each dpi, and each dpi consists of 3 individual tick samples. N, negative control ticks; P, undiluted FeLV stock solution.