Sensitivity of Lyme Borreliosis Spirochetes to Serum Complement of Regular Zoo Animals: Potential Reservoir Competence of Some Exotic Vertebrates

Introduction

Spirochetes from the Borrelia burgdorferi sensu lato (s.l.) complex, the cause of Lyme borreliosis (LB), are maintained in nature by ticks transmitting the pathogen from one vertebrate host to another. Some vertebrate hosts serve as blood meal sources for tick vectors only, while the others can be reservoir hosts for pathogens (Humair and Gern 2000). Efficient reservoir hosts for B. burgdorferi s.l. share some common characteristics: they are abundant in specific areas and serve as hosts to multiple tick vectors; they are naturally infected, remain infected often for a lifetime, and are infective to other vertebrates via competent tick vectors; and they usually do not become resistant to repeated tick feedings (Brown and Lane 1992, Oliver 1996, Gern 2008). The importance of animals as reservoirs for spirochetes is poorly described (Humair and Gern 2000, Gern 2008), and the impact of medium sized and large mammals is not yet clearly understood.

The global expansion of spirochetes is enhanced by the involvement of multiple phylogenetically diverse migratory animals; recent transoceanic migration and appearance of recombinant genotypes of pathogens; climate-related factors; recent urbanization; and an increasing overlap of human and Borrelia habitats (Rudenko et al. 2011, Rudenko et al. 2013). Changes in pathogen–host–environment interactions encourage pathogens to find new ways of exploiting novel host resources, either in domestic or wild animals. Changes that lead to increased contact among humans, domestic animals, and wildlife might lead to transmission of pathogens from an established reservoir population to a novel host population (Engering et al. 2013).

The sensitivity of spirochetes to host serum complement is a key factor in the ecology of B. burgdorferi s.l., and LB and plays a crucial role in the dynamics of global transmission of spirochetes (Kurtenbach et al. 1998, Lane and Quistad 1998). Interaction between spirochetes and host complement takes place directly in ticks during blood feeding and results in the destruction of spirochetes in the tick gut before transferring to another host. Complement sensitivity or resistance of Borrelia species is one aspect that rules Borrelia–host relationships, which is a perfect model to study the contribution of vertebrate hosts to pathogen demographic processes and to examine the interplay between the ecology of the host and the epidemiology of the bacteria (Margos et al. 2011, Movila et al. 2012).

Local, regional, and national zoos are parts of the human urban habitat. They are unique places where the human population closely interacts with wildlife and domestic animals concentrated in a restricted area. Knowledge about the reservoir capacity of zoo animals for specific pathogens of medical importance is essential to control the risk of disease spreading, especially in highly populated regions. Our recent study confirmed that zoo animals are exposed to ticks (Sirmarova et al. 2014). Here we present the results of the first expanded in vitro study of borreliacidal effects of the alternative complement pathway in the blood of exotic vertebrate species housed in five Czech zoos to the major causes of LB in Europe, B. burgdorferi sensu stricto (s.s.), B. garinii, and B. afzelii. Complement sensitivity demonstrated in our experiments might contribute to prediction of specific spirochete–reservoir host associations.

Materials and Methods

A total of 135 serum samples from 70 animal species were collected in five zoos located in different regions of the Czech Republic. Blood samples had not been specifically taken for purposes of the presented study, but for different veterinary reasons, following zoo ethics regulations. Blood samples were centrifuged at 2,500 × g for 15 min. Sera were collected, filtered through 0.22 μm polyethersulfone membrane filters (TPP Techno Plastic Products AG, Trasadingen, Switzerland), and stored at −20°C until analysis.

LYMETOP + Vet test (Promevet, Milano, Italy) was used to detect the total antibodies produced against B. burgdorferi s.s., B. afzelii, and B. garinii in animal sera. This commercial kit does not rely on a secondary antibody and is equally effective for all analyzed species (Sirmarova et al. 2014).

Eleven local B. burgdorferi s.l. strains isolated from Ixodes ricinus ticks were tested by PCR with primers designed to target the ospA gene of B. burgdorferi s.s. (set GI, 543 bp amplicon), B. garinii (set GII, 344 bp amplicon) and B. afzelii (set GIII, 189 bp amplicon) (Demaerschalck et al. 1995) to confirm the presence of single spirochete species in the culture.

Additional PCR tests were conducted to confirm the presence of pncA, vlsE, and adeC genes known to be responsible for spirochete infectivity in mammalian hosts (Radolf et al. 2012). Three main plasmids that carry those genes—lp25 (Purser et al. 2003), lp28-1 (Bankhead and Chaconas 2007), and lp36 (Jewett et al. 2007)—are crucial for the ability of spirochete to penetrate into the host body and can be lost during in vitro cultivation. Primer sets that target pncA at lp25, vlsE at lp28-1, and adeC at lp36 are conservative for all three spirochete species used in this study (R. Rego, unpublished results). Based on the results of both PCR tests, three local B. burgdorferi s.l. strains were selected for complement sensitivity testing. Selected strains were proven to contain a single spirochete species according to the results of species-specific PCR with GI, GII, and GIII primers, and each confirmed the presence of the three above-mentioned plasmids producing the pncA, vlsE, and adeC amplicons in gene-specific PCRs.

Complement sensitivity assays were conducted according to earlier published protocols (Kurtenbach et al. 1998, Bhide et al. 2005) with minor modifications. Briefly, Borrelia cultures were grown at 34°C without antibiotics to a density of 10⁷ cells/mL. Only sera samples that were negative for the presence of B. burgdorferi s.l. antibodies according to the LYMETOP + Vet test were used. Complement sensitivity tests were prepared by mixing equal amounts of serum and Borrelia suspensions to a final volume of 100 μL. All samples were incubated for 6 h at 34°C. After incubation, the cultures were examined with a dark-field microscope (Van Dam et al. 1997). Flow cytometry was used for differentiating live and dead spirochetes. Cultures were centrifuged at 8,000 × g for 10 min at room temperature. The cells were diluted with 200 μL of 2% BSA and 5.4 mM glucose in phosphate-buffered saline. Two microliters of propidium iodide (500 μg/mL) were added to each sample. The measurement was done in 5 mL round-bottom BD Falcon^™ (Fisher Scientific, Pittsburg, PA) tubes with a BD FACS Canto II flow cytometer; 30,000 events were measured. Spirochetes were differentiated from BSK-II and serum particles adjusting the fluorescence intensity and side scatter parameters. The fluorescence intensity of propidium iodide was measured in the PE-Texas Red-616/23 A channel.

Results

Results from the LYMETOP + Vet test showed that 78 out of 135 initially collected serum samples were positive for anti-Borrelia antibodies. Fifty-seven antibody-negative samples from 36 vertebrate species were included in the complement sensitivity test (Table 1). Complement sensitivity of Borrelia genospecies measured by flow cytometer correlated with the results obtained by dark field microscopy. Two controls—heat inactivated and EDTA-supplemented sera—confirmed elimination of borreliacidal effect, showing that the alternative pathway of the complement system, which operates independently of antibodies, plays a key role in spirochete killing.

All animal species included in our study could be divided into two groups based on the assay results: vertebrates that reveal no spirochete specificity in either killing or tolerating of Borrelia, and animals with spirochete-species preference (Table 2).

Sera of European rabbit, Burmese python, and radiated tortoise, as well as of some large ungulates (Table 2, numbers 1–8), revealed apparently the strongest borreliacidal effects to all tested spirochete species. The following group of even-toed ungulates that includes giraffes, oryx, antelope, and waterbuck (Table 2, nos.9–18) was highly resistant to B. garinii and B. afzelii infection with a moderate effect to B. burgdorferi s.s., killing from 50% to 80% of spirochetes in a sensitivity test. The effects of sheep, gazelle, and rhinoceros sera (Table 2, nos.19–29) on spirochetes was obviously weaker, showing the moderate bacteriolysis of borrelia. While both sheep species (Table 2, nos.19–20) were strongly resistant to B. garinii infection, the effect of sera from the rest of the animals in this group (Table 2, 21–29) on different spirochete species was rather sporadic and did not show any permanent pattern. The most diverged effect was observed in the group of seven black rhinoceros samples (Table 2, nos.23–24, 25, 26–27, 28–29). We were not able to find any age- or sex-related explanations of such paradoxes, and the speculation that rhinoceros responses to LB spirochetes are rather individual requires analysis of the wider group of samples.

The subsequent group of ungulate samples (Table 2, nos. 30–35) revealed evidence of spirochete species preference. It is obvious that both zebra and cattle species as well as gorals are tolerant to B. burgdorferi s.s. spirochetes and resistant to B. garinii and B. afzelii. Black wildebeest and Asian lion sera (Table 2, nos. 36–38) revealed strong borreliacidal activity against B. garinii and high tolerance to B. burgdorferi s.s. and B. afzelii. The same pattern is observed in humans. However, the moderate effect of human sera against B. garinii rather suggests a tolerance to the infection with this species. The same conclusion can be drawn for Barbary sheep, taking into consideration the weak to moderate bacteriolysis capacity of it sera against all three spirochete species (Table 2, 41).

Defassa waterbuck as well as both bird species revealed the highest resistance to B. afzelii and the high tolerance to B. burgdorferi s.s. and B. garinii, which was expected for birds, but not for ungulate (Table 2, 42–45). The rest of samples, which includes carnivores, even-toed ungulates, and rodent and reptiles species (Table 2, 46–57) showed no borreliacidal effect against B. burgdorferi s.s., B. garinii, or B. afzelii.

With one exception—the group of black rhinoceros samples—the rest of the vertebrate species showed rather definite patterns in their response to infection: those that are resistant to all three analyzed spirochete species (Table 2, 1–22) and those that are tolerant to them (Table 2, 46–57). With regard to spirochete species preference, the majority of analyzed vertebrate species are tolerant in different degrees to either “generalist” B. burgdorferi s.s. (Table 2, 9–22 and 30–35), or to it and one of the “specialists” species, B. garinii or B. afzelii, (Table 2, 36–45).

Discussion

In nature, all animal species included in our study serve as hosts for multiple species of ixodid ticks, primarily from genera Amblyomma, Hyalomma, Rhipicephalus, Haemaphysalis, Dermacentor, and Ixodes (Horak et al. 1991, Horak et al. 2010). The vast majority of vertebrates serve as carriers or reservoir hosts of a remarkable array of tick-borne pathogens of medical and veterinary importance, including protozoa, viruses, and bacteria that might affect public health and have severe implications for domestic animals (Junker et al. 2015, Mackenstedt et al. 2015). The significance of vector–host interactions in the transmission of arthropod-borne disease agents has been recognized. Pharmacological activity of tick saliva can have a profound effect on pathogen transmission (Nuttall et al. 2000). The salivary glands of ticks provide numerous anti-inflammatory, anti-haemostatic, and anti-immune molecules that, for example, control histamine, bind immunoglobulins, or inhibit the alternative complement cascade providing a privileged site at the tick–host interface in which Borrelia and other tick-borne pathogens are sheltered from the normal innate and acquired host immune mechanisms that fight infections (Nuttall et al. 2000). The interaction between spirochetes with complement has a major impact on spirochete transmission dynamics. Complement-mediated selection operates directly in the midgut of the feeding tick, so spirochetes are destroyed directly in tick, prior to transmission to the host. Vertebrate host range for Borrelia is determined by spirochetes sensitivity to complement of particular animal species; the ability to bind factor H and FHL-1 appears to depend on the Borrelia genotype (Kurtenbach et al. 2002, Stevenson et al. 2002). Vertebrate hosts may be infected concurrently with different genospecies of B. burgdorferi s.l., even with those for which they are apparently not transmission competent. A possible explanation for this obvious paradox may be related to the fact that Borrelia genes are differentially expressed during the life cycle. The gene expression of spirochetes residing in the tick's midgut resembles the one observed in culture when the major outer surface proteins of Borrelia, including possible complement receptors, appear to be down-regulated. Studies to identify complement receptors on the spirochetes and to unveil the protective mechanisms of spirochetes against complement represent one of the fascinating areas in complete understanding of three-way interactions among spirochetes, the tick, and the host (Tsao 2009).

Recent urbanization drastically changed the dynamics between wild and domestic animal species and the human population. As a result, some wild species (foxes, hairs, hedgehogs, raccoons, wild cats, deer, wild boars, etc.) are attracted to peri-urban and urban areas due to abundance of food and shelter (Mackenstedt et al. 2015). Exotic wild species are transferred from the areas of their natural habitat to urban landscapes, changing the local composition of vertebrate hosts important for the maintenance of ticks and spreading of zoonotic pathogens in restricted areas of city zoos, parks, and landscape gardens. Here we present the results of the first extended in vitro analysis of sensitivity of LB spirochetes to serum complement of exotic animals housed in zoos.

Transmission of vector-borne pathogens between wildlife and domestic animals is an issue of major interest due to the potential for spreading infectious diseases to humans. Zoos are a unique environment for the interaction of exotic and native vertebrates, arthropod vectors, and human populations (Lau et al. 1994, Sirmarova et al. 2014). Our previous studies showed that zoo animals kept in open-fenced areas are exposed to arthropod vectors of multiple pathogens, including tick-borne encephalitis virus and LB spirochetes (Sirmarova et al. 2014). While the reservoir capacity of rodents and birds for B. burgdorferi s.l. is well studied (Craine et al. 1997, Kurtenbach et al. 1998, Huegli et al. 2002, Kurtenbach et al. 2002, Poupon et al. 2006, Gern 2008, Mannelli et al. 2012), the role of large animals, such as ungulates, is not yet properly defined. We did not plan to evaluate the borreliacidal capacity of zoo animals' complement in exact numbers, but to define the pattern of Borrelia sensitivity to serum of exotic species common in central European zoos.

Wild and domestic ungulates were not considered as competent reservoir hosts for Borrelia; however, they serve as hosts for a large number of ticks, significantly contributing to the vector population (Mannelli et al. 2012). The strong borreliacidal effects to B. burgdorferi s.s., B. garinii, and B. afzelii were previously revealed in large ungulates such as cows, deer, and cattle (Kurtenbach et al. 1998, Kurtenbach et al. 2002). Contrary to that, our results showed that other species, such as African buffalo, nyala, and warthog, did not reveal any borreliacidal effect to studied spirochete species. We confirmed the tolerance of other cattle species, wildebeest, gorals, and gazelle to B. burgdorferi s.s. It is obvious that the low reservoir competence defined for a few ungulate species (Randolph et al. 1996) should not be applied to all ungulates in general, as this group consists of quite diverse vertebrate species. Most probably, different ungulate species exhibit a different reservoir capacity. However, tolerance to spirochetes does not necessarily make a vertebrate a competent reservoir host. All species that we analyzed are native to Africa, where the presence of spirochetes from B. burgdorferi s.l. complex has not yet been confirmed. However, being habitants of zoos located in highly endemic European LB areas, they could contribute to spirochete transmission by tick vectors.

Tolerance of both zebra species in our project to B. burgdorferi s.s. is in agreement with earlier results (Kurtenbach et al. 1998) that showed that horse serum had borreliacidal effects to B. garinii, B. valaisiana, B. afzelii, and B. japonica but not to B. burgdorferi s.s., confirming that Equidae are capable as reservoir hosts for this species.

Resistance of spirochete species to wild feline serum in our research is consistent with earlier finding based on experiments with domestic cat serum, suggesting a role of these animals in the circulation of LB spirochetes between hosts and vectors (Bhide et al. 2005).

The difference between the complement system of mammals and ectothermic animals, including reptiles, is significant. Several studies have described the complement components and the complement-mediated killing of B. burgdorferi s.s. in selected reptile species (Koppenheffer 1987, Lane and Quistad 1998, Kuo et al. 2000). Blood from western fence lizard (Sceloporus occidentalis) and Southern alligator lizard (Elgaria multicarinata) was borreliacidal to B. burgdorferi s.s., while American anoles (Anolis carolinensis) and southeastern five-lined skinks (Eumeces inexpectatus) could serve as reservoirs for this species (Levin et al. 1996). In our study, turtle serum showed strong borreliacidal effect to all studied Borrelia species, a finding supported by data from Koppenheffer (1986), who demonstrated the presence of classical and alternative pathways in turtle serum. The same strong borreliacidal ability was revealed in sera of Burmese python.

The presence of antibacterial activities in American alligator was confirmed (Zimmerman et al. 2010). Similarly, evidence for a potent and broad-acting complement system in the serum of Siamese crocodile revealed its high efficiency against Salmonella typhi, Escherichia coli, Staphylococcus aureus, S. epidermidis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Vibrio chorelae (Preecharram et al. 2008, Zimmerman et al. 2010, Kommanee et al. 2012). Contrary to this we found no boreliacidal effect to any Borrelia species in Siamese crocodile's serum. The Siamese crocodile is a highly endangered freshwater reptile species native to exotic Asian countries. This species vanished from many geographic regions of its previous distribution. The high tolerance of Siamese crocodile for LB spirochetes is a fact of scientific value and interest. However, the impact of this species in the spirochete enzootic cycle in Europe is most probably insignificant.

Representation of bird samples in our study is very low. Nevertheless, it is evident that neither flamingo nor ostrich can be infected with B. afzelii. It is highly possible that B. burgdorferi s.s. will survive in birds, but certainly B. garinii is the primary species that use birds as reservoir hosts (Table 2).

Killing of pathogens is the main task of host innate immunity. Host complement plays the crucial role in this process. Avoiding the killing by complement is the main goal of invading Borrelia that employs differential expression of genes encoding surface proteins, including surface exposed complement regulator-acquiring surface proteins (CRASPs), in its survival. Borrelia CRASP expression correlates with its resistance to host serum (Kreizcy et al. 2001, Blom et al. 2009). It is possible that deeper analysis of this complex opposition can lead to identification of new candidate(s) for anti-Borrelia vaccines.

Conclusions

Results on the sensitivity of three LB spirochete species, B. burgdorferi s.s., B. garinii, and B. afzelii, to serum complement of exotic zoo animals showed that (a) resistance of Borrelia to host serum complement is species-specific and applies to both spirochetes and the hosts; (b) different hosts possess different tolerance to LB spirochetes that might vary among the individuals of the same genera or species and is not affected by age or sex of the host; (c) carnivores possess apparently the highest reservoir competency for LB spirochetes; (d) selected ungulate species are tolerant to presence of Borrelia infection and might serve as reservoir hosts for B. burgdorferi s.l.; and (e) B. burgdorferi s.s. showed the highest ability to infect a wider spectrum of host species, confirming its “generalist” status.