Transstadial and transovarial perpetuations of Borrelia venezuelensis in Ornithodoros rudis, with demonstration of vector competence

Abstract

Ticks of the genus Ornithodoros are recognized vectors of relapsing fever group Borrelia, such as Borrelia venezuelensis, whose infection dynamics in its vector, Ornithodoros rudis, remains poorly understood in Brazil. This study aimed to investigate the transstadial perpetuation and transovarial transmission of B. venezuelensis in O. rudis and the vector competence of all parasitic stages of this tick species. Experimental colonies were maintained under controlled laboratory conditions, and ticks were fed on Syrian hamsters (Mesocricetus auratus). Infection in hamsters was monitored using dark field microscopy and real-time PCR. Borrelia venezuelensis was detected across multiple tick life stages, and its presence in larvae derived from infected females confirmed transovarial transmission. Vector competence for B. venezuelensis was demonstrated for larvae, nymphs and adults of O. rudis. Additionally, neurological symptoms and sudden death were observed in some hamsters, possibly associated with toxicosis due to heavy tick infestation. These findings provide the first experimental evidence of transstadial and transovarial maintenance of B. venezuelensis in O. rudis, reinforcing its role as a competent vector and contributing to a better understanding of the eco-epidemiology of relapsing fever in South America.

Introduction

Tick vector competence, defined as the ability to acquire, maintain, and transmit pathogens, is influenced by factors such as host-seeking behavior, duration of host contact, transstadial perpetuation, and transovarial transmission of the pathogen (Vial 2009; Sonenshine and Roe 2014). For transmission to be effective, the pathogen must replicate sufficiently within the vertebrate host to reach a burden that enables uptake by the vector. Additionally, rapid adaptation to the vector’s internal environment, which differs physiologically and immunologically from that of the vertebrate host, is essential for the pathogen’s survival and perpetuation (Boyle et al. 2014; Estrada-Peña et al. 2018; Lopez et al. 2021). Transstadial perpetuation and transovarial transmission are critical mechanisms for the maintenance of microorganisms within vector populations across multiple generations, representing key strategies employed by various pathogens. Therefore, understanding the interactions among the mammalian host, the pathogen, and the vector is essential for the development of effective strategies to control vector-borne diseases (Neelakanta et al. 2016; Embers et al. 2019; Lopez et al. 2021).

Tick-borne relapsing fever is a disease caused by bacterial agents of the genus Borrelia phylogenetically assigned to the relapsing fever group (RFG). Except for one species (Borrelia recurrentis), which is transmitted by the human body lice, all RFG Borrelia species are transmitted by ticks (Trevisan et al. 2021). Although there are a few Borrelia species associated with hard ticks (Ixodidae family), most of the tick-borne RFG borreliae are associated with soft ticks (Argasidae family), chiefly of the genus Ornithodoros (Trevisan et al. 2021).

Most of the tick-borne RFG borreliae are zoonotic agents that circulate between vertebrate animals and soft ticks in different regions of the world, causing human disease (relapsing fever) when infected ticks bite humans (Cutler 2015). Despite the widespread occurrence of tick-borne RFG borreliosis in temperate and tropical regions of the world (Talagrand-Reboul et al. 2018; Trevisan et al. 2021), the disease has been notably neglected in South America (Faccini-Martínez et al. 2022). Human cases of tick-borne RFG borrelial infectione have been reported in Colombia and Venezuela, with all the reports restricted to the first half of the 20th century. These South American cases, together with others reported in Panama during the same period, were attributed to the RFG agent Borrelia venezuelensis transmitted by its vector tick Ornithodoros rudis (Faccini-Martínez et al. 2022).

It was only recently that O. rudis was confirmed in Brazil; previous reports cast doubt on its presence in the country (Dantas-Torres et al. 2009). During field work in 2017, Muñoz-Leal et al. (2018) collected 30 specimens of O. rudis (25 adults and 5 nymphs) from debris of a bird nest inside a hollow palm-tree at Riachão Municipality, Maranhão State, northeastern Brazil. Attempting to isolate tick-borne spirochetes, the authors fed the adult ticks (10 males, 15 females) on naïve vesper mice (Callomys callosus). One animal, upon which a female O. rudis was fed, showed motile spirochetes in blood using dark-field microscopy (Muñoz-Leal et al. 2018). The spirochetemic blood was further processed by in vitro isolation (Muñoz-Leal et al. 2018); the genome of the borrelial isolate was published as B. venezuelensis (Kneubehl et al. 2022).

The incrimination of O. rudis as a vector of B. venezuelensis was based on studies carried out in Colombia, Panama and Venezuela, in which spirochetemia was demonstrated in laboratory mammals (mice, rats) and in a human, after inoculation with field-collected body homogenates of O. rudis (Dunn 1927; Dunn and Clark 1933; Pifano et al. 1941). In one of these studies (Dunn and Clark 1933), vector competence was demonstrated by exposing a human volunteer to infestations by several stages of O. rudis ticks that had been collected from a household where human cases of relapsing fever had been reported. In addition, O. rudis first instar-nymphs (N1) transmitted B. venezuelensis to a monkey (Macaccus rhesus) after larval-acquisition feeding on a spirochetemic rat, demonstrating transstadial perpetuation of the agent from larva to N1 nymph, and the vector competence of the latter (Dunn and Clark 1933). More recently, vector competence was demonstrated for a field-collected O. rudis female from Brazil, which transmitted B. venezuelensis to a vesper mouse in the laboratory (Muñoz-Leal et al. 2018). To our knowledge, no further studies on vector competence or transstadial perpetuation have been reported for B. venezuelensis, for which the transovarial transmission remains untested.

In the present study, we used the colony of O. rudis from the study of Muñoz-Leal et al. (2018) to evaluate transovarial and transstadial perpetuation of B. venezuelensis, and the vector competence of larvae, nymphs, and adults of O. rudis from Brazil.

Materials and methods

In the study of Muñoz-Leal et al. (2018), field-collected O. rudis adults (15 females and 10 males) were allowed to feed on tick-naïve vesper mice. After feeding, some of the 15 females (generation F₀) produced offspring, which were all pooled and used to establish a laboratory colony. The F₁ offspring were reared to F₁ adults by feeding on tick-naïve hamsters (Mesocricetus auratus), which were not tested for borrelial infection after the feeding of each tick stage. After this initial procedure, the present study started with 56 F₁ adult ticks (22 males, 34 females).

The 56 F₁ adult ticks (22 males, 34 females) were allowed to feed to repletion on a hamster. After that, the offspring of these females were pooled (F₂ larvae) and reared to the adult stage (F₂ adults) by feeding the ticks of each stage (larvae, nymphs, or adults) or each nymphal instar (N1, N2 and N3) on a single hamster. Thereafter, the F₃ offspring were reared to the adult stage by feeding F₃ larvae, the three nymphal instars (N1, N2 and N3) and adults, each on a single hamster (Fig. 1).

Fig. 1.

Diagram illustrating experimental procedures with the laboratory colony of Ornithodoros rudis ticks from F₁ adults to F₃ adults. Each tick stage/instar from one generation fed on a single hamster (numbered from H1 to H11). Tick off-host development (molting, oviposition and egg incubation) occurred in an incubator at 26 °C and 80% relative humidity

For tick feeding, each hamster was chemically anesthetized using an intramuscular injection of a combination of ketamine (80 mg/kg) and xylazine (10 mg/kg). After sedation, the ventral region of each rodent was shaved to facilitate tick attachment. Ticks were placed in a plastic feeding chamber (Levin and Schumacher 2016) and allowed to feed until full engorgement (20–60 min). Upon detachment, fully engorged ticks were transferred to labeled plastic tubes (6 cm long, 4 cm wide) containing paper to absorb humidity and maintained in an incubator.

Off-host developmental stages of the ticks were maintained in an incubator under controlled conditions of 26 °C, 80% relative humidity, and 24 h-scotophase/day. All the hamsters used in this study came from a laboratory animal room, with no previous contact with ticks or tick-borne agents. Hamsters were used for feeding a single tick stage/instar and generation with no hamster used for more than one infestation. Before infestation with ticks, a drop of blood (2.5 µl, measured with a micropipette) was obtained from each hamster by ear vein-puncture, expressed onto glass slides, and visually monitored by dark-field microscopy to detect the presence of motile spirochetes, as previously described (Oliveira et al. 2023). This procedure was repeated daily up to 21 days after infestation (DAI). When detected, the total number of spirochetes per sampling day was calculated by summing the number of motile spirochetes counted in a total of 20 microscope fields at 200× magnification.

From each infested hamster, a blood sample (50–100 µl) was collected between 12 and 15 DAI through eye capillary puncture, after chemical anesthesia as described above. DNA was extracted from hamster blood using the PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Hamster blood DNA was tested by a Borrelia-specific real-time PCR assay using primers Bor16S3F and Bor16S3R (and an internal probe Bor16S3P) targeting a 148 bp fragment of the Borrelia 16 S rRNA gene, as described (Parola et al. 2011). To confirm that the detected borreliae was B. venezuelensis, some samples positive by real-time PCR were tested by a conventional PCR assay targeting a 354-bp fragment of the borrelial flaB gene (Stromdahl et al. 2003). PCR products were sequenced and submitted for BLAST analysis (www.ncbi.nlm.nih.gov/blast) to determine the closest identities available in GenBank. In all PCR assays, Borrelia anserina DNA and water were used as positive and negative controls, respectively.

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Veterinary Medicine of the University of São Paulo (protocol number 2458021221).

Results

A total of 11 hamsters (numbered from H1 to H11) were used, one for feeding each tick stage/instar from one generation, from F₁ adults to F₃ adults (Table 1). During the 21-day course of dark-field microscopy monitoring of hamsters, motile spirochetes were visualized in blood several days after tick infestations on hamsters H1, H2, H5, H6, and H7, as detailed in Table 1. Hamster H3 and H4 died at 5 and 7 DAI, respectively, without spirochetes been observed in blood. These two hamsters developed neurological disorders (ataxia, tremors) and died suddenly with generalized paralysis. The spleen of hamster H3 was collected and tested by the real-time PCR assay, but no Borrelia DNA was detected. Hamster H4 was not molecularly tested.

Table 1.

Data on infestations of 11 hamsters (H1 to H11) with different stages of a laboratory colony of Ornithodoros rudis that was previously shown to be naturally infected with Borrelia venezuelensis. Each infested hamster has its blood daily examined for the presence of motile spirochetes by dark field microscopy from 0 to 21 days after infestation (DAI)

Hamster no.	Tick Generation	Tick stage/instar	No. ticks that fed on the hamster	DAI that motile spirochetes were visualized in hamster blood
H1	F1	Adults	56	5 to 14
H2	F2	Larvae	≈ 600	7 to 13
H3	F2	Nymphs N1	300	none*
H4	F2	Nymphs N2	220	none*
H5	F2	Nymphs N3	120	10 to 16
H6	F2	Adults	30	11, 13 to 17
H7	F3	Larvae	≈ 600	14 to 18
H8	F3	Nymphs N1	117	none
H9	F3	Nymphs N2	88	none
H10	F3	Nymphs N3	52	none
H11	F3	Adults	30	none

*Hamsters H3 and H4 died at 5 and 7, respectively, days after infestation with ticks

Transmission of borreliae was observed in hamster H1 (infested with F₁ adults), indicating that the O. rudis colony remained infected by B. venezuelensis from the F₀ adult female reported by Muñoz-Leal et al. (2018). Thereafter, F₂ larvae successfully transmitted borreliae to hamster H2, indicating that B. venezuelensis was transmitted transovarially from F₁ females to F₂ larvae. Transmissions of B. venezuelensis by N1 and N2 nymphs (F₂) could not be demonstrated because the two infested hamsters (H3 and H4) died (Table 1). The subsequent stage, N3, successfully transmitted borreliae to hamster H5, as did the F₂ adult ticks to hamster H6. The F₃ larvae successfully transmitted borreliae to hamster H7, indicating that B. venezuelensis was transmitted transovarially from F₂ females to F₃ larvae. However, borrelial transmission to hamsters could not be demonstrated in any of the subsequent stages/instars of F₃ ticks (nymphs N1 to adults feeding on hamsters H8 to H11) (Table 1).

When motile spirochetes were detected in hamster blood, the number of spirochetes counted in a total of 20 microscopic fields varied from 1 to 22 per day, with maximum counts at 10 to 12 DAI for hamsters H1, H2 and H5, 13–14 DAI for H6, and 16–17 DAI for H7 (Fig. 2). Borrelial DNA was detected in hamster blood by real-time PCR at 12 DPI in hamsters H1 and H2, and at 15 DPI in hamsters H5, H6 and H7. No borrelial DNA was detected in the blood collected at 15 DPI from hamsters H8 to H11. Blood from hamsters H6 and H7 were tested by conventional PCR and sequencing, which generated a flaB partial sequence that was 100% identical (300/300 bp, after removal of primer sequences from the flanks) to the GenBank flaB sequence of B. venezuelensis (MG651650).

Fig. 2.

Results of dark-field examination of blood samples of hamsters H1, H2, H5, H6 and H7 on days after infestation with Ornithodoros rudis. Values represent the total number of motile spirochetes per 20 microscope fields at 200x magnification on each sampled day

Discussion

This study provides experimental demonstration of transstadial perpetuation of B. venezuelensis among all feeding stages of O. rudis for three generations. Previous studies with O. rudis demonstrated limited transstadial perpetuation, from larva to the first nymphal instar only (Dunn and Clark 1933). Here we demonstrated transovarial transmission of B. venezuelensis during two consecutive generations of O. rudis. Although transovarial transmission of RFG borreliae may be a common feature in Ornithodoros spp. (Schwan and Raffel 2021), results have varied. For instance, it was successfully demonstrated for Borrelia crocidurae in Ornithodoros erraticus but not in Ornithodoros savignyi (Gaber et al. 1984), whereas transovarial transmission of Borrelia duttonii in Ornithodoros moubata varied among different borrelial strains (Tabuchi et al. 2008). Successful transovarial transmission in the present study might be related to using a population of O. rudis that was naturally infected with B. venezuelensis.

Vector competence, as determined by the ability to transmit B. venezuelensis, was demonstrated for the three feeding stages (larvae, nymphs and adults) of O. rudis. These findings complement previous studies that demonstrated transmission competence for N1 nymphs (Dunn and Clark 1933), a female tick (Muñoz-Leal et al. 2018) and pooled feeding stages (Dunn and Clark 1933). Surprisingly, after transmission and maintenance of the borrelial infection throughout all tick developmental stages from F₁ adults to F₃ larvae, transmission of B. venezuelensis was not demonstrated for F₃ post-larval stages. This result could be related to low infection rates by B. venezuelensis in F₁ and F₂ ticks. Because the infestations with F₃ nymphs consisted of much lower number of individuals than in the infestations with F₂ nymphs, it is possible that the infection by B. venezuelensis was lost in F₃ post-larval stages. This statement is based on the possible low infection rate of the field collected adults, for which Muñoz-Leal et al. (2018) demonstrated transmission of B. venezuelensis to mice for only one (4%) out of 25 field-collected adults. Alternatively, it is possible that the F₃ nymphs still harboured a very low load of spirochaetes, which was insufficient to cause infection in their hosts. Unfortunately, these ticks were not molecularly tested to confirm this possibility.

The two hamsters that died of paralysis (H3 and H4) were precisely those that were infested with a greater number of nymphs, three to five times higher than the numbers of nymphs used in the other hamsters in this study. The absence of borrelial DNA amplification in the blood and organs of one of the hamsters indicates that the death was not related to infection with B. venezuelensis. In a study with Ornithodoros aff. puertoricensis, Venzal et al. (2007) observed that mice exposed to higher tick loads showed nervous incoordination (ataxia and tremors) from 5 DAI, dying by the 8th DAI. They ruled out the role of any tick-borne pathogen and concluded that the animals died of tick toxicosis. Since the clinical picture presented by hamsters H3 and H4 was similar, it is possible that they also died of tick toxicosis due to high tick load.

From an ecological and epidemiological perspective, the results of this study reinforce the relevance of O. rudis as a competent vector of B. venezuelensis and suggest that this tick species may also act as a reservoir for the bacterium in natural environments, contributing to its persistence even in the absence of infected vertebrate hosts. This is particularly relevant in settings such as caves, nests, or shelters of wild animals, where argasid tick density can be high and human contact is occasional, yet sufficient to trigger sporadic outbreaks of relapsing fever. Moreover, this study provides a solid experimental foundation for future research aimed at developing surveillance, control, and prevention strategies for Borrelia-associated relapsing fever in Brazil. The confirmation of vector competence and the ability of O. rudis to maintain the pathogen may support public health policies grounded in the One Health approach, integrating veterinary, environmental, and human health actions.

Conclusions

This study demonstrated, for the first time, the transstadial perpetuation among all tick developmental stages and transovarial transmission of B. venezuelensis in O. rudis. The infection was maintained across multiple tick developmental stages and transmission to vertebrate hosts, providing strong evidence of vector competence. These findings underscore the importance of argasid ticks in the maintenance of pathogenic Borrelia species and expand our understanding of the ecology of tick-borne diseases.

Author contributions

FRJ conceptualized the study, conducted all experiments, performed data analysis, and wrote the main manuscript text. MPM assisted with sample collection and data analysis. ISS contributed to sample collection and data analysis. GMBO assisted with sample collection and interpretation of results. SML assisted in the experimental design, collected samples, and contributed to data interpretation. MBL supervised the project and contributed to the experimental design, data interpretation, and manuscript preparation. All authors reviewed and approved the final manuscript.

Funding

The Article Processing Charge (APC) for the publication of this research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (ROR identifier: 00x0ma614).

Data availability

We declare all data is being provided within this manuscript.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.