Detection of Antibodies Against Anaplasma phagocytophilum in Algerian Mice ( Mus spretus ),Portugal

Abstract

The recent detection of Anaplasma phagocytophilum in Portugal stimulated further research on the agent's enzootic cycle, which usually involves rodents. Thus a total 322 rodents belonging to five species, including 30 Apodemus sylvaticus (wood mouse), 65 Mus musculus (house mouse), 194 M. spretus (algerian mouse), 5 Rattus norvegicus (brown rat) and 28 R. rattus (black rat), were studied by indirect immunofluorescent assay (IFA) and/or polymerase chain reaction (PCR) for A. phagocytophilum exposure in four sampling areas of mainland and two areas of Madeira Island, Portugal. Overall, 3.6% (7/194) of M. spretus presented with IFA-positive results. Seropositive mice were detected in all three mainland sampling areas where this species was captured, with prevalence of 5.2% (5/96) and 5.0% (1/20) for the Ixodes-areas of Arrábida and Mafra, and 1.3% (1/78) for Mértola, a difference that was not statistically significant (p > 0.05). The majority of IFA-positive mice were detected in spring when considering either Arrábida alone (p = 0.026) or all M. spretus sampling areas together (p = 0.021), although the significance of this association was not evident after Bonferroni correction. Nevertheless, neither the seropositive M. spretus, nor additional samples of 10% seronegative rodents from mainland, and 16% of rodents collected in Madeira Island showed evidence of A. phagocytophilum active infections when spleen and/or lung samples were tested by PCR. Either the M. spretus results represents residual antibodies from past A. phagocytophilum infections, present infections with limited bacteremia, or cross-reactions with closely related agents deserves more investigation.

Introduction

R odents are important hosts for several Ixodes ticks (Acari:Ixodidae), especially for larvae, to some extent also for nymphs, and in the case of host-specific species, for adults as well (Zeidner et al., 2000; Bown et al., 2003, 2006). Thus, it is not surprising that rodents play a role in enzootic cycles of several Ixodes-borne agents, such as the human pathogens Borrelia burgdorferi s.l., Babesia spp. and tick-borne encephalitis (TBE) viruses.

Anaplasma phagocytophilum [formerly Ehrlichia phagocytophilum, E. equi and human granulocytic ehrlichiosis (HGE) agent, (Dumler et al., 2001)] is another example of an Ixodes-borne agent. This worldwide bacterium has long been known in the veterinary field as a cause of disease in ruminants, horses, dogs and cats, but it is also regarded as an emerging human pathogen with increasing importance in both the United States and Europe (Strle, 2004; Dumler, 2005). Despite the broad-range of susceptible hosts, the occurrence of A. phagocytophilum variant strains with distinct pathogenicity and enzootic cycles has been suggested. Both molecular and experimental cross-infection studies has demonstrated that variant strains associated to human, equine, and canine disease are identical or highly similar but distinct from those associated with ruminants (Chae et al., 2000; Pusterla et al., 2001; Stuen et al., 2003). Variant strains have been detected in ticks but the apparent lack of A. phagocytophilum transovarial transmission in these arthropods indicates that competent vertebrates, especially those that represent a feeding support for larvae and nymphs, play potential roles in the maintenance of agent's active cycles. Although the natural history of A. phagocytophilum is still unfolding, variant strains causing disease in cows and sheep are especially found in wild ruminants such as cervids (Massung et al., 2005, 2006). In contrast, the maintenance of those involved in non-ruminant disease is believed to be dependent on tick-rodent cycles (Massung et al., 2003), with humans and possibly domestic animals being involved as incidental “dead-end” hosts. In the US Peromyscus leucopus and Neotoma fuscipes are competent reservoirs in Eastern and Western regions, respectively (Telford et al., 1996; Castro et al., 2001; Foley et al., 2002). Moreover, PCR-based studies show A. phagocytophilum DNA in several other rodent species, including Myodes (formerly Clethrionomys) gapperi, Neotoma mexicana, Microtus orchogaster, Peromyscus maniculatus, P. truei, Sciurus griseus, Spermophilus lateralis, Tamias minimus and T. striatus in US (Walls et al., 1997; Nicholson et al., 1999; Zeidner et al., 2000; DeNatale et al., 2002; Lane et al., 2005), Apodemus agrarius, A. flavicollis, A. sylvaticus, Myodes (formerly Cletrionomys) glareolus, Microtus agrestis, M. oeconomus and Rattus rattus in Europe (Liz et al., 2000; Bown et al., 2003, 2006; Christova & Gladnishka, 2005; Grzesczuk et al., 2006), and Apodemus agrarius, A. peninsulae and Tamias sibiricus in Asia (Cao et al., 2006).

In Portugal, A. phagocytophilum DNA has been detected in two Ixodes species, Ixodes ricinus collected from Madeira Island and I. ventalloi on the mainland (Santos et al., 2004). Moreover, the Public Health importance of Portuguese strains of A. phagocytophilum is under study given the recent detection of active infection in a horse (Santos, 2007) and given the presence of antibodies against this agent in Ixodes-exposed patients (Santos et al., 2006). Although no information is available regarding the natural history of A. phagocytophilum in Portugal, several rodents might be involved in agent's maintenance as they are known hosts for Ixodes species that can sustain active infection, including Apodemus sylvaticus, Elyomis quercinus, Mus spretus, Rattus norvegicus for both I. ricinus and I. ventalloi, and also Rattus rattus and Sciurus vulgaris for I. ricinus (Dias et al., 1994; MM Santos-Silva, personal communication). Based on serological and molecular analyses of archived rodent sera and organ samples available from Center for Vector and Infectious Diseases Research, National Institute of Health Dr. Ricardo Jorge (CEVDI/INSA), the present study investigates the potential role of Portuguese rodent species in the maintenance A. phagocytophilum enzootic cycles.

Material and Methods

Sample collection

Rodents were captured from 1998 through 1999 on Madeira Island (Santana, Seixal) and from 1999 through 2004 in three littoral areas (Gerês, Arrábida, Mafra) and one inland area (Mértola) of mainland Portugal. Ixodes ticks are commonly found in Madeira Island and the above mentioned littoral areas of mainland, henceforth designated in this work as Ixodes-areas. Santana and Seixal are on the north coast of Madeira Island and rodents were captured in peridomestic sites and farm fields lining the urban perimeter. Madeira Island is characterized by deep ravines and gorges and a mildly subtropical climate. Gerês is located in the north mainland and rodents were collected in an oak forest of the Peneda-Gerês National Park. This area has cold winters, abundant rainfall and high humidity. Mafra and Arrábida are located in the center of the country. In Mafra, rodents were captured in the protected dense forested Park of Tapada Nacional de Mafra. Climate here is temperate and humid with moderate precipitation. The Arrábida trapping site was in an abandoned farming field with ruderal vegetation located near a wooded area. Summers here are hot and dry and winters are cold and humid. Mértola is located in the south of Portugal and trapping were performed in an orange tree orchard and near an artificial pond with the vegetation consisting mainly of rushes. Here the summer is hot and dry and there is little rainfall even in winter. The animals were captured as part of ongoing projects to study population dynamics and rodent-borne diseases by using baited Sherman and Tomahawk live-traps (H.B. Sherman Traps, Inc., Tallahassee, Florida; Tomahawk Live Traps Company, Tomahawk, Wisconsin). One trapping session was performed per season on the mainland, and only in spring on Madeira Island, each conducted over three consecutive nights. The methodological differences observed between areas of study are due to the retrospective nature of this work. In fact, the rodent samples obtained from the CEVDI/INSA archives were gathered in behalf of different projects, thus with different objectives and methodologies. Trapping and handling procedures were approved by the Institute for Conservation of Nature and Biodiversity (ICNB) and performed according to the Directive 86/609/EEC. Once in the laboratory, rodents were anesthetized and biometric measurements were recorded to aid in species identification. Whole-blood samples were collected in serum-gel microtainers (Microtainer; Becton Dickinson, New Jersey) by submandibular venous plexus puncture and sera were recovered either by gravity sedimentation or by centrifugation at 1000 × g for 10 min, and saved for serology. After euthanasia by cervical dislocation, organs were aseptically harvested, in most cases including spleen and lung samples (mainland specimens) or only lung samples (Madeira Island specimens); samples were individually cryopreserved in liquid nitrogen until use.

Serology

For detection of A. phagocytophilum antibodies, an indirect immunofluorescent assay (IFA) was used to screen all the available rodent sera. This included only mainland specimens, since no sera were available from Madeira Island rodents. Briefly, sera were diluted at 1:40 in PBS and incubated on antigen slides prepared with A. phagocytophilum Webster strain-infected HL-60 cells. Serum obtained from both naïve and A. phagocytophilum inoculated laboratory-reared mice were included on each antigen slide as controls. A second incubation with fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin G (IgG) (Dako Cytomation, Denmark) or anti-rat IgG (Serotec, UK) in an optimal dilution of 1:40 in PBS containing 1:6000 Evans blue was used to identify bound antibodies. For the purpose of this study an IFA titer ≥40 was interpreted as positive. All positive samples were serial diluted to determine the end-point titer, which was reported as the reciprocal of the highest dilution at which specific fluorescence is observed.

Molecular analysis

Organ samples from all seropositive and 10% of seronegative rodents captured on mainland and 16% of rodents obtained from Madeira Island were tested for A. phagocytophilum DNA by polymerase chain reaction (PCR). Genomic DNA was extracted from spleen (10 mg) and lung (20 mg) samples following the Qiamp Tissue Kit procedure (Qiagen GmbH, Germany). Samples from naïve laboratory-reared mice A. phagocytophilum were included in every group of ten extractions as controls. Molecular analysis was performed by nested or single tube PCR reactions using different sets of primers: (i) MSP465f/MSP980r derived from the highly conserved 5′ region of major surface protein-2 (msp2) paralogous genes (Caspersen et al., 2002); (ii) GE3a/GE10r followed by GE9f/GE2 which amplify a of a 546-bp fragment of the 16S rRNA gene (rrs) (Massung et al., 1998); (iii) HS1/HS6 followed by HS43/HS45 for the amplification of a 442-bp sequence of heat-shock operon, groESL (Sumner et al., 1997). Primer sequences and amplification conditions were previously described (Sumner et al., 1997; Massung et al., 1998; Caspersen et al., 2002). PCR was performed in a total volume of 50 μl, containing 1 μM of each primer, 2.5 U of Taq DNA polymerase and 200 μM of each deoxynucleotide triphosphate and 50 mM KCl 10 mM Tris-HCL pH 8.3, and 1.5 mM Mg²⁺ (Eppendorf MasterTaq Kit, Germany), and 10 μl DNA extract (or 1 μl of amplicon in nested reactions). Water and DNA from known negative samples and A. phagocytophilum Webster strain genomic DNA were tested in each PCR run as controls. Prevention of cross-contamination was managed by using barrier pipette tips and by performing PCR in a separate room from that used for DNA extraction.

Statistical analysis

The independence of proportions of positive rodents was compared by a two-sided Fisher's exact test using R-project for Statistical Computing version R-2.6.2 (available under a GNU public license at http://www.r-project.com). An alpha level of 0.05 was selected to indicate statistical significance. The Bonferroni correction of alpha was used for multiple testing.

Results

A total of 322 rodents belonging to five species in four genera were included in this study, comprising 30 Apodemus sylvaticus, 65 Mus musculus, 194 M. spretus, 5 Rattus norvegicus and 28 R. rattus. The distribution of rodent species per sampling area in mainland Portugal and Madeira Island are presented in Figure 1.

FIG. 1.

Number of A. phagocytophilum seropositive cases per total number of rodent studied by IFA and/or PCR, according to sampling area (trapping period).

The 294 rodents captured on mainland, including 30 A. sylvaticus, 42 M. musculus, 194 M. spretus and 28 R. rattus, were initially screened by IFA for the presence of A. phagocytophilum antibodies. Overall, 7 M. spretus were found to be seropositive, presenting IFA titers ranging from 80 (n = 6) to 320 (n = 1). Seropositive M. spretus were detected in all three sampling areas where this species was captured, including Arrábida, Mafra and Mértola. The highest seroprevalences were observed in the Ixodes-areas of Arrábida and Mafra (n = 5 [5.2%] and n = 1 [5%], respectively), although the difference was not statistically significant when compared to Mértola (n = 1 [1.3%]; p > 0.05 for all comparisons) (Table 1). Moreover, M. spretus were not more likely to have A. phagocytophilum antibodies (p = 0.099) than the other species and no difference was observed regarding gender (p > 0.05 for all comparisons). However, analysis of proportions showed an association between spring and seropositive mice when considering either Arrábida alone (p = 0.026) or all M. spretus sampling areas together (p = 0.021). Yet, the statistical significance of these results was not evidenced after the significance level correction for multiple testing (p > 0.0125, by Bonferroni correction).

Table 1.

Seroprevalence of A. phagocytophilum in M. spretus Mice Captured in Mainland, Portugal

	No. (%) IFA-positive/No. tested
Sampling area	Arrábida	Mafra	Mértola	All areas
Gender:
Male	2(3.3)/60	1(8.3)/12	1(2.1)/47	4(3.4)/119
Female	3(8.3)/36	0/8	0/31	3(4.0)/75
Capture season:
Spring	3(20.0)/15^*	1(8.3)/12	0/8	4(11.4)/35^*
Summer	1(2.9)/34	—	0/21	1(1.8)/55
Autumn	0/29	0/8	1(12.5)/8	1(2.2)/45
Winter	1(5.6)/18	—	0/41	1(1.7)/59
Total	5 (5.2)/96	1(5.0)/20	1(1.3)/78	7(3.6)/194

A dash denotes trapping that has resulted in no captures

p < 0.05

Spleen and lung samples from all 7 IFA-positive M. spretus were tested by PCR for the presence of A. phagocytophilum DNA, but no positive results were obtained. PCR results were also negative for an additional 29 samples (95% CI 0-12%) randomly selected from seronegative rodents, representing 10% of the captures performed in each mainland sampling area, including: 2 A. sylvaticus from Gerês; 4 M. musculus and 2 M. spretus from Mafra; 9 M. spretus and 1 R. rattus from Arrábida; 1 A. sylvaticus, 8 M. spretus and 2 R. rattus from Mértola.

The 28 rodents randomly selected from the captures performed in Madeira Island, including 23 M. musculus and 5 R. norvegicus, were tested only by PCR, since no sera samples were available at CEVDI/INSA for serology. PCRs performed on lung samples were negative for the three A. phagocytophilum genes analyzed.

Discussion

This study provides evidence for exposure of M. spretus to A. phagocytophilum or a closely related agent in Portugal. Seropositive mice were detected in all sampling areas where this species was captured, including the Ixodes-areas of Arrábida and Mafra, as well as Mértola. Of interest is that collection sites in Arrábida (in a 0.5 ha farm) were located in the same valley and 2 km straight-line from the area defined as Baixa de Palmela (Setúbal District) where a previous PCR-based study detected A. phagocytophilum infections in I. ventalloi ticks (Santos et al., 2004). Moreover, M. spretus is a known vertebrate host for both I. ventalloi and I. ricinus ticks (Dias et al., 1994); the latter tick species is regarded as the major vector of A. phagocytophilum in Southern, Central and Northern Europe (Strle, 2004).

In general, M. spretus mice can be found across the country especially in grassland, arable land, and rural gardens, selecting for humid biotypes. This mouse is usually crepuscular to nocturnal and is maximally active during spring through summer. The period from spring to early summer is also regarded as the time when most of A. phagocytophilum infections or reinfections occur in rodents, a seasonality that possibly results from the abundance of Ixodes nymphs, and has been demonstrated in multicapture studies by examining PCR and/or by examining increase in specific antibody titers of rodent populations (Stafford et al., 1999; Castro et al., 2001). In our study, spring was indeed the season in which the majority of A. phagocytophilum-positive mice were detected but this association was found to be non-significant after the conservative Bonferroni correction. However, the p-values obtained nearly reached the borderline significance level which is highly suggestive, but probably due to the limited number of animals studied not strong enough to be provide statistical evidence.

In areas where A. phagocytophilum cycles are known to occur, a proportion of rodents are simultaneously PCR and IFA-positive, varying from 20-68% for P. leucopus (Walls et al., 1997; Yeh et al., 1997; Stafford et al., 1999; Levin et al., 2002), 58-100% for N. fuscipes (Nicholson et al., 1999; Castro et al., 2001; Foley et al., 2002) and 20-33% for P. truei (Nicholson et al., 1999; Castro et al., 2001). These findings seem to reflect either the occurrence of persistent infections or the interval during which immunity is strengthening before the elimination or suppression of bacteremia. Most of the studies regarding seasonal dynamics of A. phagocytophilum showed that although the majority of infections appeared to be transient, in a limited number of rodents, the agent can persist. Active infections of A. phagocytophilum can last at least one month in naturally-infected M. glareolus (Bown et al., 2003), 60 days-14 months in naturally and experimentally infected N. fuscipes (Castro et al., 2001; Foley et al., 2002), and 1-10 months in naturally and experimentally infected P. leucopus (Stafford et al., 1999; Levin et al., 2002; Massung et al., 2004). Additionally, Levin and Fish (2000) showed that seropositive P. leucopus are only partially protected from reinfection. Regardless, in our case no A. phagocytophilum IFA-positive M. spretus had an active infection, even using a cut-off criteria between 1:16-1:80 as the referenced studies (Walls et al., 1997; Yeh et al., 1997; Stafford et al., 1999; Castro et al., 2001; Nicholson et al., 2001; Foley et al., 2002; Levin et al., 2002). The analysis of low antibody titers is essential, particularly when little is known and clinical signs of illness cannot be discerned (as often the case in wildlife), although the risk is false positive serological reactions that could bias seroprevalence results. As a compromise for sensitivity and specificity, all sera were screened at 1:40, a dilution below the cut-off value currently used in our laboratory for human serology that is broadly accepted as the minimal definition of seropositivity against A. phagocytophilum (Dumler et al., 1995). In fact, all samples that were found reactive in the initial dilution also had detectable antibody at a dilutions equal to or above 1:80. Moreover, molecular analyses were performed using PCR protocols generally considered to be highly sensitive (Sumner et al., 1997; Massung et al., 1998; Caspersen et al., 2002) and using spleen samples, widely accepted as adequate for detection of A. phagocytophilum infection in both natural and experimentally-infected rodents (Liz et al., 2000; Martin et al., 2000). PCR results were also negative for an additional sample comprising 10% of all seronegative rodents randomly selected in each mainland sampling area.

The presence of residual antibodies could result from past infections or active A. phagocytophilum infection with low level bacteremia that is undetectable by current methods. In fact, inefficient transient infections in mice were described in an experimental study using Ap-variant 1, a dominant A. phagocytophilum genotype that seems important in ruminant disease (Massung et al., 2003). A. phagocytophilum identified in Portuguese ticks seems distinct from Ap-variant 1, and at least for those detected in I. ricinus sequences similar to non-ruminant disease strains were observed. These data imply that infection of mice and other small mammals is likely important in natural maintenance of A. phagocytophilum in Portugal. Besides rodents, both I. ricinus and I. ventalloi may parasitize several other small and medium size mammalian that could be potential reservoirs for A. phagocytophilum, including members of the orders Soricomorpha, Lagomorpha, Erinaceomorpha, and Carnivora (Dias et al., 1994; MM Santos-Silva personal communication). Moreover, the potential presence of A. phagocytophilum ruminant strains in our country can not be excluded because no study has yet addressed this animal population. A Spanish study performed on I. ricinus collected from bovines identified the presence of Ap-variant 1, and this proximity is motivation for further study (Portillo et al., 2005). Apart from the economic relevance of A. phagocytophilum ruminant strains, a new and interesting epidemiological context for these strains has been recently suggested. This concept argues that A. phagocytophilum ruminant strains could interfere or compete with the non-ruminant strains for tick niches, perhaps influencing the incidence or prevalence of human and non-ruminant animal infections (Massung et al., 2002, 2003).

Finally, it is also important to consider the possibility of cross-reactions with agents that share antigenic similarities with A. phagocytophilum. For example active infections by the closely related A. platys were recently detected in Portuguese dogs stimulating serologic reactions against A. phagocytophilum (Santos et al., in press a). Although, other details regarding the enzootic cycle of A. platys are largely unknown active infections in rodents have been already documented in other countries (Chae et al., 2003; Kim et al., 2006). However this does not seems to be the case since both the groESL and rrs primers are known to amplify A. platys DNA (Sumner et al., 1997; Massung et al., 1998).

A percentage of rodents from the other Ixodes-area on Madeira Island were also studied by PCR for the presence of A. phagocytophilum infections, but no positive results were obtained. R. norvegicus and M. musculus were the only species captured in the two sampling areas of Santana and Seixal, despite the fact that the rodent fauna of this Island also includes R. rattus (Almeida, 1996). To date, no report has yet described R. norvegicus as a potential reservoir for A. phagocytophilum. Regarding the peridomestic M. musculus, several inbred strains derived from this species are frequently used in experimental infections with A. phagocytophilum (Borjesson & Barthold, 2002). It is also important to note that lungs were the only available sample from Madeira Island rodents, and lack of positive results in this tissue should be carefully interpreted. This organ is less frequently used for PCR testing than spleen, although an experimental study performed by Martin et al. (2000) suggests its potential utility to detect A. phagocytophilum infections. The presence of A. phagocytophilum in Madeira Island has been demonstrated by the detection of infected I. ricinus at different periods of time (Núncio et al., 2000; Santos et al., 2004). Yet whether the agent's cycles are supported by rodents or other vertebrate animals remains unknown. This Island presents a great variety of fauna that could serve as hosts for I. ricinus, including several bird species, bats, ferrets, goats, sheep, cows, horses, dogs, and cats (domestic and feral) (Almeida, 1996). Interestingly, the molecular analysis of infected ticks has documented the existence of an A. phagocytophilum identical or very similar to North America strains implicated in human cases of disease (Santos et al., in press b), which argues for a more detailed and directed study to investigate the agent's enzootic cycles and its potential health threat to both humans and animals in this Portuguese island.

Footnotes

Acknowledgements

This research was partially supported by the Portuguese government through the Fundação para a Ciência e a Tecnologia grant BD/8610/2002. Rodents capture was supported by Instituto Nacional de Saúde Dr. Ricardo Jorge, Fundação para a Ciência e a Tecnologia PRAXIS/PCNA/BIA/135/96 and POCTI/ESP/39549/2001.

Disclosure Statement

No competing interests exist.

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