Transovarial transmission and finding of Trypanosoma rhipicephalis in the hemolymph of Rhipicephalus sanguineus sensu latoa

Copyright Kaulich et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted non-commercial use, distribution, and reproduction in any medium provided the original work is properly cited. Transovarial transmission and finding of Trypanosoma rhipicephalis in the hemolymph of Rhipicephalus sanguineus sensu latoa


Abstract
Trypanosoma rhipicephalis is a species isolated from Rhipicephalus microplus ticks collected from native bovine Seropédica, in the state of Rio de Janeiro. This study aimed to investigate the interaction of the tick Rhipicephalus sanguineus s. l. with T. rhipicephalis by in vitro artificial feeding. Eight females of R. sanguineus s. l. partially fed on rabbits. Tick infection was performed by an artificial feeding system using plastic tips for 12 hours. Canine blood used for feeding the ticks test group was previously infected with T. rhipicephalis 10 8 /mL. The hemolymph smear test was performed in all females after experimental infection. The daily posture was collected and organized in pools of each female per posture day. The eggs were divided into three groups, the first group for eggs PCR, the second for hatching and larval PCR, and the third group of eggs for isolation in cell culture. The evaluation of the presence of DNA in the macerated eggs of experimentally infected ticks showed two positive PCR samples. The evaluation of the presence of DNA in experimentally infected females showed all samples tested positive. For the hemolymph test, tick number 7 presented epimastigote developmental forms and amastigotes of T. rhipicephalis. Experimental infection by artificial feeding proved to be a suitable tool to study the interaction of T. rhipicephalis in R. sanguineus s. l. ticks. The results show the transovarial transmission of T. rhipicephalis by R. sanguineus s. l., as well as the interaction of the protozoan in the organism of this tick species. Keywords: ticks, artificial feeding, trypanosomatids.

Introduction
Parasites of the genus Trypanosoma are unicellular and flagellated microorganisms belonging to the Trypanosomatidae family with wide geographical distribution. Hematophagous arthropods act as biological and mechanical vectors for different species of this family, infecting a wide variety of hosts, ranging from plants to animals, vertebrates and invertebrates (Hoare, 1972); (Morzaria et al., 1986).
Ticks, as blood feeders, are predisposed to the ingestion of a variety of parasites during feeding and have been proposed as vectors for Trypanosoma species (Morzaria et al., 1986); (Burgdorfer et al., 1973); and (Krige et al., 2019). Artificial tick feeding is an important tool for studying the transmission of pathogens in the absence of vertebrate hosts (Chabaud, 1950); (Valim et al., 2017). Cell culture of tick embryonic cells is an important tool for studying the interaction between cells of these arthropods and pathogens transmitted by them because it may help define the complex nature of the host-vector-pathogen relationship (Bell-Sakyi et al., 2007).
Trypanosoma rhipicephalis Marotta et al. (2018) is a species isolated from R. microplus ticks from native Seropédica cattle (Marotta et al., 2018). Little is known about its biological cycle; aspects related to possible pathogenesis and interaction with other species are unknown (Marotta et al., 2018).
The present study aims to investigate the interaction of Rhipicephalus sanguineus sensu lato and T. rhipicephalis by means of artificial feeding of ticks.

Material and methods
The experiments were performed at the Parasitic Diseases Laboratory of the Department of Epidemiology and Public Health of the Veterinary Institute (IV) of the Federal Rural University of Rio de Janeiro (UFRRJ), located in building 1 of the Veterinary Institute, in the municipality of Seropédica, Rio de Janeiro.
Eight females of R. sanguineus s. l. from the tick colony maintained at the Parasitic Disease Laboratory of the UFRRJ were partially fed on rabbits, following the methodology described by Valim et al. (2017). Two groups (G) were formed, one specimen was fed uninfected blood (G1 control), and seven were fed infected blood (G2 test). Tick infection was performed by an artificial feeding system using plastic tips ( Figure 1) for a period of 12 hours following the procedures described by Valim et al. (2017). Blood used for artificial tick feeding was collected aseptically from the dog's cephalic vein by the Vacutainer® system coupled to a 5 mL tube containing citrate anticoagulant. The material was labelled and stored under refrigeration at 4 °C for up to 24 hours.
To perform Trypanosoma blood infection, the dog's blood serum complement system was previously inactivated so as not to cause reactions with the inoculated agent. For inactivation of the complement system, collected blood was centrifuged at 5000 rpm for 10 minutes. Then, the supernatant was kept for 40 minutes in a 56 °C water bath. The pellet was resuspended with PBS in a volume equivalent to the initial blood volume. Then two washes with PBS were performed. After the serum reached room temperature (26 °C), it was homogenized with the red blood cells. Canine blood used to feed G2 test ticks was previously infected with 10 8 T. rhipicephalis/mL.
The hemolymph smear test was performed in all females after the experimental infection, being evaluated from the first DPI (the day after infection) until the posture, i.e., 13th DPI. The females used were kept frozen for later DNA extraction and PCR analysis. Hemolymph samples collected from the distal (tarsal and/or tibial) section of one or more teleogin legs were deposited on a glass slide, identified, fixed for three minutes in methanol and stained by the Giemsa 10% method for 25 minutes.
The daily posture was collected and organized in pools of each female per day of laying. Eggs were separated into three groups, the first for egg PCR, the second for hatching and larval PCR, and the third group for isolation of eggs in cell culture.
The DNA from the pool of macerated eggs, larvae, and infected females used in the experiment were extracted using the phenol-phenol/chloroform method previously described (Santolin et al., 2013). During each extraction battery, a blank sample was extracted along with the test samples as procedural control. A conventional Polymerase Chain Reaction (PCR) was performed to detect the presence of the 24S rDNA Trypanosomatid gene, following the protocol of the original article (Souto et al., 1999). Two positive samples (Trypanosoma amblyomii) and two negative samples (ultrapure water) were used during the PCR reaction.

Results
The authors were successful in infecting R. sanguineus s. l. with T. rhipicephalis, a species recently described in R. microplus by Marotta et al. (2018). The ticks R. sanguineus s. l. were infected by artificial feeding, and epimastigote and amastigote forms (Figure 2) of T. rhipicephalis were recovered from the hemolymph. The morphological forms observed were pleomorphic, such as those observed in in vitro cultures isolated from Rio de Janeiro native ticks, previously described by Marotta et al. (2018).

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Transovarial transmission and finding of Trypanosoma rhipicephalis in the hemolymph of Rhipicephalus sanguineus sensu lato Evaluation of the presence of DNA in experimentally infected females showed all positive test group samples. By hemolymph test, tick number 7 showed epimastigote evolutionary forms of T. rhipicephalis.
The evaluation of DNA presence in the macerated eggs of experimentally infected ticks showed two positive PCR samples as well as the evaluation of the presence of DNA in macerated larvae, which presented one positive sample. The macerated eggs of the control G1 did not present positive bands by PCR. The PCR positive egg pool samples were from two G2 test females, one from the 4th to 6th DPI of tick number 4 and the other from the 4th to 6th DPI of tick number 6. The PCR positive larval pool sample also belonged to tick number 6 on the 6th to 8th DPI.

Discussion
This study describes for the first time how the tick Rhipicephalus sanguineus s. l. can be infected with u T. rhipicephalis sing artificial feeding. The original description of T. rhipicephalis and preliminary studies conducted by Marotta et al. (2018) indicate its phylogenetic proximity to the KG1 species described by Thekisoe et al. (2007). Trypanosoma KG1 was isolated from naturally infected Haemaphysalis hystricis ticks in Japan (Thekisoe et al., 2007). The morphological and phylogenetic proximity between T. rhipicephalis and Trypanosoma KG1 seems to be related to the fact that it is also isolated from naturally infected ticks.
Another species with phylogenetic proximity to T. rhipicephalis is the species T. caninum isolated from an axenic culture of the intact skin of a domestic dog captured in different states of Brazil, including Rio de Janeiro (Madeira et al., 2014). Although the T. caninum vector is not yet known, it is possible that it is transmitted by ticks which then justifies the phylogenetic proximity between T. rhipicephalis and T. caninum. T. rhipicephalis has not been found to infect or cause any evidence of clinical signs in animals; there is a possibility that T. rhipicephalis has bovines as a mammalian host because tick R. microplus infests this species preferentially.
Although most Trypanosoma species are transmitted by hematophagous insects, ticks also appear to be likely vectors of some species of this genus (Madeira et al., 2014), (Kaufer et al., 2017), (Krige et al., 2019). In Brazil, a trypanosomatid with morphological characteristics similar to Trypanosoma theileri is naturally found in R. microplus and reported as nonpathogenic for cattle and usually transmitted by tabanids (Martins et al., 2008), (Ribeiro et al., 1988;Brum et al., 2012).
The T. rhipicephalis species in the present study showed biological characteristics compatible with other protozoa of the genus Trypanosoma, and evolutionary forms without visible free flagella in axenic cultures were seen. Recently, Barros et al. (2015) described aflagellar epimastigote forms in T. caninum confirmed by electron microscopy.
These results suggest that R. sanguineus s. l. may interact with the agent, considering its presence in the hemolymph. The present study also suggests the possible transovarial transmission of the protozoan by the tick R. sanguineus s. l. Transovarian transmission is a very significant mechanism for maintaining T. rhipicephalis in the environment and says a lot about its propagation and maintenance in the next generation of ticks.
R. sanguineus is a three-host life cycle species and is a competent disseminator of disease-causing microorganisms between infected and uninfected animal hosts (Dantas-Torres et al., 2013); (Sonenshine & Roe, 2014). The one-host tick R. microplus also takes three blood meals; however, feeding is restricted to the same host, making parasites ingested by one-host tick dependent on transovarial transmission for successful circulation (Kahl, 2018).
This study contributes a research tool for future studies on the possible elucidation of the T. rhipicephalis biological cycle and the involvement of the tick R. sanguineus s. l.

Conclusion
Experimental infection through artificial feeding proved to be an adequate tool to study the interaction of T. rhipicephalis in R. sanguineus s. l. The results indicate a possible transovarial transmission of T. rhipicephalis by R. sanguineus s. l., as well as its interaction with the protozoan.