Role of the Lizard Teira dugesii as a Potential Host for Ixodes ricinus Tick-Borne Pathogens

Abstract

PCR screening of ticks and tissue samples collected from 151 Teira dugesii lizards seems to indicate a potential role of this lizard species in the maintenance and transmission cycle of some Ixodes ricinus tick-borne agents, such as Rickettsia monacensis, Rickettsia helvetica, and Borrelia lusitaniae, that are circulating on Madeira Island.

TEXT

Lizards have been identified as vertebrate hosts of ixodid ticks (3). The existence of an endemic lizard population of Teira dugesii species that have become one of the most prevalent vertebrate species on Madeira Island and are known to host immature stages of Ixodes ricinus drew our attention. Additionally, on this island, I. ricinus has been shown to be infected with different genospecies of Borrelia spp., Rickettsia monacensis, and Anaplasma phagocytophilum (6, 10, 11, 17). A comprehensive investigation was thus undertaken to study the potential role of T. dugesii in the maintenance and transmission of I. ricinus tick-borne agents.

One hundred fifty-one tissue samples from lizard tails and 211 I. ricinus ticks removed from 100 lizards were collected monthly from March 2009 to February 2010 in the Calheta and Campanario areas on Madeira Island. DNA samples were analyzed by PCR for Rickettsia, Borrelia, and A. phagocytophilum using specific primer pairs targeting two genes for each agent, as previously described (2, 4, 9, 16, 19). Additionally, 60 DNA samples extracted from lizard blood prints were tested for A. phagocytophilum.

Ticks were present only in lizards captured in Calheta (n = 130) and were mostly found in the forelimb axial region. An average of 2.1 ticks per animal was observed. Nymphs were detected in all studied periods, but larvae were present only from early April to November (Fig. 1).

Fig 1.

Numbers of lizards and their ticks (nymphs and larvae) collected in Calheta.

Lizards and ticks infected with Borrelia lusitaniae, R. monacensis, Rickettsia helvetica, and A. phagocytophilum were found only in Calheta, as shown in Table 1. Rickettsia-positive amplicons showed 100% sequence identity with gltA (341/341 bp) and ompB (384/384 bp) of R. helvetica (GenBank accession numbers U59723 and HQ232251) and 100% identity with gltA (341/341 bp) and ompB (384/384 bp) of R. monacensis (GenBank accession numbers HM149283 and EU883092), previously detected in Portuguese ticks (6, 12). Two main genetic variants of B. lusitaniae were detected in the intergenic spacer (IGS) partial sequence from 5S (rrf) to 23S (rrl): variant 1, presenting 100% identity to PotiB6, a tick isolate previously reported on Madeira Island (GenBank accession number EU078961) (6), and variant 2, detected in only one tick and showing 100% identity to PoAnB1, a Portuguese isolate from Apodemus sylvaticus (GenBank accession number EF647595) (7). A. phagocytophilum-positive amplicons showed 100% sequence identity to previously described rrs sequences from Portuguese ticks (GenBank accession number EU098006) (16). However, groESL gene analysis revealed two main polymorphic positions in the encoding sequence, enabling the identification of three genetic variants: variant A, found in one lizard and eight ticks and showing 100% identity to a North American strain isolated from humans (GenBank accession number U96728), variant B (nucleotide 401, G→A; nucleotide 497, T→C), detected in five ticks, and variant C (nucleotide 401, G→A), found in two ticks and 100% identical to the A. phagocytophilum partial sequence detected in an I. ricinus species from Madeira Island (GenBank accession number EU004826) (17).

Table 1.

Prevalence of Borrelia burgdorferi sensu lato, Rickettsia spp., and Anaplasma phagocytophilum infection in lizard tissue samples and ticks collected from lizards

Sample type and stage (n)	No. (%) infected with:					Coinfection(s) (no. [%])a
	Rickettsia monacensis	Rickettsia helvetica	R. monacensis or R. helvetica	Borrelia lusitaniae	Anaplasma phagocytophilum
Ticks
Larvae (65)	20 (30.8)	0	20 (30.8)	3 (4.6)	0	1 (1.5) Bl + Rm
Nymphs (146)	67 (45.9)	2 (1.4)	69 (47.3)	22 (15.0)	15 (10.3)	10 (6.8) Bl + Rm; 5 (3.4) Ap + Rm
Total (211)	87 (41.2)	2 (1.4)	89 (42.2)	25 (11.8)	15 (7.1)	16 (7.6)
Lizard tissues
Adults (83)	6 (7.2)		6 (7.2)	3 (3.6)		0
Juveniles (68)	4 (5.9)	2 (1.3)	6 (7.2)	4 (5.9)	1 (0.7)	0
Total (151)	10 (6.6)	2 (1.3)	12 (7.9)	7 (4.6)	1 (0.7)	0
Lizard blood prints
Total					0

^aBl + Rm, Borrelia lusitaniae and Rickettsia monacensis coinfection; Ap + Rm, Anaplasma phagocytophilum and Rickettsia monacensis coinfection.

Rickettsia was the most prevalent agent detected in both lizards and ticks, with a higher infection in nymphs. An interesting finding was the presence of R. monacensis and R. helvetica in lizard tissues because the latter had never been detected on the island. These results suggest not only that disseminated infections can occur in T. dugesii but also that this species may be a potential or transitory reservoir infecting ticks. The fact that all the ticks collected from lizards were localized on the lizard forelimb axial area and that sample tissue for PCR detection was removed from the tail seems to indicate that disseminated infection may occur. R. monacensis had previously been detected by PCR on Madeira ticks, albeit with a lower prevalence (20%) than that of the present study (41.1%) (6). This may be due to the screening of different areas and the fact that ticks were collected in vegetation and not in lizards. The detection of B. lusitaniae in lizard tissues and the high level of infected parasitizing ticks also support the importance of T. dugesii in the maintenance of this genospecies on Madeira Island, a finding which is in agreement with those of studies carried out in other lizard species (1, 5, 8, 15). B. lusitaniae has also been associated with other reservoirs, such as rodents (A. sylvaticus) and migratory birds (7, 14). It is suggested by several authors that bird ticks may be responsible for the geographic dispersion of B. lusitaniae, and both rodents and lizards may be responsible for perpetuating this genospecies in enzootic cycles in which they are the major vertebrate hosts for I. ricinus (7, 15). As described for R. monacensis, a higher prevalence (11.8%) of B. lusitaniae in ticks from lizards was found in this work than in our previous work (2.7%) on Madeira Island (6). Another important finding in this study is the presence of polymorphisms that reveal the presence of genetic variants of B. lusitaniae, which appear to be specifically associated with lizard hosts, in some sequences. Further studies need to address the eventual evolution forces responsible for this genetic diversity and the presence of B. lusitaniae subpopulations associated with specific reservoirs. Other Borrelia genospecies, such as Borrelia afzelii, Borrelia garinii, and Borrelia valaisiana, that were previously detected in ticks on Madeira Island in the same area were not detected in either ticks attached to lizards or lizard tissues (11). Currently, we are testing the hypothesis that T. dugesii might have a borrelicidal effect on some Borrelia genospecies other than B. lusitaniae.

Regarding A. phagocytophilum, the data obtained thus far suggest that Madeira Island lizards are exposed to infected ticks but that they do not seem to be a primary reservoir host for this agent. In fact, the sole T. dugesii positive tail snip detected contrasts with the number of studied lizards and with the much higher number of positive parasitizing nymphs found in the same area. Moreover, no DNA was found in the lizard blood prints tested, even with the blood-borne nature of this agent. Since A. phagocytophilum is not transovarially transmitted, the absence of A. phagocytophilum infection in all attached larvae suggests that lizards were not infectious for ticks and raises the hypothesis that positive nymphs may have resulted from previous infections as larvae when feeding on other reservoir hosts. These results are in accordance with previous findings in an experimental infection study of A. phagocytophilum in lizards (13). Studies performed by Matuschka and collaborators (10, 11) suggest that rats are potential reservoir hosts for Borrelia on this island. This could also be true for A. phagocytophilum. Nevertheless, a preliminary study performed on Madeira Island in rodents did not find A. phagocytophilum infection in two of the three species present on the island (18). During the present study, field work was also directed toward the capture of rodents, but unfortunately, no animals were captured, a result which might be related to recent massive campaigns for rodent control in the area.

In conclusion, Madeira Island harbors a large population of T. dugesii lizards that seem to be involved in the maintenance and transmission cycle of at least two tick-borne agents. Future experimental studies involving R. monacensis maintenance in lizards and its transmission to feeding ticks are crucial to evaluate whether lizards are transitory reservoirs and how long they can sustain the infection. The study of other potential hosts is also important to understand the complex interaction between ticks and hosts and pathogens on this island.